Team:GreatBay SCIE/Human Practices
Integrated HP
Intro
Breast Cancer is one of the most prominent cancer presents in females. In 2020, 11.7% of the newly diagnosed cancer cases are breast cancers, the largest among the 36 cancer. Among women, 24.5% of the cancer cases were breast cancer, and 15.5% of death is caused by breast cancer as well[1]. Stage I breast cancer can be cured with surgical operations; surgeries involve the removal of breast and lymph nodes, in some cases, some chest muscles have to be removed as well. In later stages, chemotherapy and radiotherapy has to be applied as well. However, Chemotherapy and radiotherapy are harmful to the body, causing problems such as falling hair. Targeted drugs remain flawed in many ways, for example, antibody-drug conjugates often cause great side effects; pathway inhibitor drugs rely on specific pathways which sometimes can be bypassed by tumor cells.
Understanding the Problem: Clinical & Laboratory Methods
Clinical
We wish to understand breast cancer not only from statistical perspective, but also from the doctors and the patients; this could help us understand breast cancer in a more practical approach.
Highlights:
To understand how breast cancer is treated clinically, we interviewed doctors who are breasts cancer specialists and also with a breast cancer screen company. In these interviews, we discussed the limitations of current treatments and also their view of good treatments.
Interviewing with the doctors we talked about targeting drugs: they are expensive in price, still have issues with stability and specificity. In addition, we talked about surgeries: Mastectomy and Lumpectomy. Lumpectomy (Breast-conserving surgeries) is considered to be healthier for the patients but it is not promoted in China, with a breast-preserving rate of only 22% compared to that of 80% in western HICs*. This is much due to lack of facilities and conserving mindset of many doctors.
The worst problem of all is lack of knowledge of breast cancer, only 5% of the population in China screens for breast cancer which caused most cases to be discovered during late stages, which is harder to treat.
Interview with Doctors, Breast Cancer Specialists:
Dr. Nong, from Peking University Shenzhen Hospital.
Dr. Zhou, from Shenzhen Nanshan Hospital
Dr.Lu, from Xinhua Hospital.
When asked about treatments for breast cancer, Dr Nong spoke to us about monoclonal antibodies and ADC drugs. They are the two most frequently used drugs to treat HER2-positive breast cancer. She also revealed that the cost of targeted drugs is expensive, especially for certain imported drugs. The most commonly used monoclonal antibody is Trastuzumab. But according to Dr. Nong, the price of Trastuzumab is high, with approximately 5500 RMB each dose. Hoping to explore more about the cost of Her-2 positive breast cancer treatments, we connected with Dr Zhou. She told us that Her2-positive patients who need targeted drugs might need 200,000 RMB in total, which is a significant amount of cost for general families. She also claimed that although antibody-conjugate drugs exhibited great effectiveness, there are certain limitations. Large molecules like antibodies have immunogenicity which are likely to trigger an unwanted immune response in bodies. Moreover, along with the transport, there is a possibility that drugs are disassociated with the antibody, which might accidentally kill normal cells. Dr. Nong expressed her high expectations for a drug delivery system with high specificity and stability.
Besides drugs, we've also talked about surgeries: Mastectomy and Lumpectomy (breast-conserving surgery). Doctor Lu gave our team an online lecture about these two surgeries. In 2018, in breast cancer treatment, the proportion of breast-conserving surgery in China was only 22%, far lower than that of 80% in European countries as well as in the US. We also learned that doctors tend to avoid mastectomy which may result in appearance defects for females. Breast cancer doctors will seek the best way to do breast-conserving surgeries. However, these types of surgeries are coupled with radiotherapy to ensure all the breast cancer cells are killed. But things aren't always done this way, doctors that are more conservative in many Chinese second or third-tier cities are more likely to take Mastectomy surgery is their first choice. Moreover, some small hospitals cannot provide sufficient equipment for radiotherapy, so they cannot perform breast-conserving surgeries. From another perspective of things, we learned that patients generally pay little attention to breast health from the doctors: they rarely check-in hospitals and are not aware of self-checking at home, which makes breast cancer a substantial potential risk for people. Overall, this lecture gave us an idea that hospitals and communities should promote breast-conserving surgeries in China.
Although there are many treatments for breast cancer, all three doctors claimed that ' early screening and early treatment is the best 'cure' for cancer. Breast cancer screening was free, but most people are unaware of it and don't check regularly.
From the interview with the doctors, we could grasp much information that would later help us identify the problem. (See Problems Identified🔍)
Aisono, a breast cancer screening company.
From the information given by the doctors, we were curious and interviewed a breast cancer screening company.
From our talk, they told us that in China, only 5% of the female goes for breast cancer screening and in Shenzhen, only 150 thousand out of the 20 million people screens for breast cancer. Some part of it is due to a lack of sonographers registered in China: only 140,000, which is far from enough to provide screening for the general public in China.
This is where Aisono found opportunity and market, they developed machines to automate the process of breast cancer screening, using artificial intelligent to analyze sonographs to be able to spot tumors during early development.
"We believed that the future of public health lies in prevention, not in treatment."
This idea also inspired our Public engagement part of the project. (see Problems Identified🔍)
Laboratory
Other than surgeries, drugs and chemicals play an important part in treating breast cancer. Drugs however, aren't developed in hospital beds, they are tested in labs by researchers and scientists. So we started a series of interviews and discussion to talk about the research aspect of treating breast cancer.
Highlights
In order to develop and formulate our project, we interview specialists to learn about pharmacy research.
Limitations of current drug treatments lie in the level of toxicity and mark>specificity. We learned about how Antibody drugs tackle these issues and slowly formulated our project. We also interviewed pharmaceutical companies to learn about the process to validate our design, this includes in vitro tests using cancer cell lines. From a broader perspective, we learned that most pharmaceutical companies aim at improving existing platforms of drug development and few choose to develop innovative drugs.
Dr. Qiu
We interviewed Dr. Qiu (Clay Qiu), who is currently working in a pharmaceutical company in Suzhou, and specializes in areas accociating with antibodies drugs.
From Dr. Qiu, we learned that there are currently 4 types of targeting therapy for HER2 positive breast cancer (H2BC).
1. Kinase inhibitors
2. Antibody drugs
3. Antibody Drug Conjugates
4. CAR-T Therapy
(See more in Description)
We also discussed the relationship between drug toxicity and targeting ability of a targeting drug. Dr. Qiu told us that, as for a cancer targeting drug, stronger (more toxic) does not mean better. The more you do harm to the tumor, the more you do harm to the body. Many ADC type drugs were halted during clinical phase because their side-effects were too strong.
"We have to strike a balance, that is, how to avoid killing normal tissue cells while killing cancer cells as much as possible"
We also learned that Doxorubicin and Maytansine are two types of chemicals used in targeting drugs.
Furthermore, we continued our discussion on current obstacles in treating breast cancer. Dr. Qiu stated that, even for HER2-positive cancer patients, not all cancer cells overexpress HER2, thus killing only the HER2-positive cells might not be as effective we think.
We also asked Dr. Qiu about how drugs could remain intact during circulation inside the body, a factor to consider mentioned by our instructor Dr. Zong. Dr. Qiu told us that past targeting drugs, uses antibody which is a stable factor present in the body. An example is people using the FC domain of the antibody in ADC drugs, by binding chemicals onto this domain, their high affinity for each other would allow the drug to leave by exoplasmosis after being endocytosed by the normal tissue cells.
At last we asked the question:
What are the characteristics of the ideal cancer-treating drug?
Dr. Qiu's answer is summarized in the following:
"First is high specificity, this is the basis for increased toxicity of the drug. However, after decades of research in chemotherapy, scientists have tried many drugs and chemicals but so far we can conclude that monotherapies are just not enough to cure cancer; the micro-environment of the tumor tissue is too large to neglect drug resistance. Now, people are trying out combined methods, immunotherapies, and other therapies to compensate for the drawbacks of many treatments."
(Dr. Qiu also guided us during our idealation and laboratory stage, which you can find in Problems Identified🔍 & Ideate and Design.)
Pharmaceutical companies
We also interviewed several pharmaceutical companies, to understand the current state of drugs as well as the process of drug production.
Innovent
Through our communication with Innovent, we learned that only a few of the company paid attention to developing new anti-cancer drugs in China. Most of the companies focused on chemistry manufacture and control, which is called the CMC department. This department utilizes existing drug producing methods and expands the quantity and quality within the existing platformed protocol, modified them to obtain the larger yields, higher quality controllable products.
Company N (Due to privacy agreements, we cannot show their name)
By talking with their consultant, we learned that there are two types of drugs: innovative and biosimilar products. Biosimilar products compete for lower prices, while innovative drugs compete for effectiveness.
We were also informed of the stages during drug development.
Pre-clinical, phase 1, phase 2 and phase 3. Which is used to test out the effectiveness and side effects of the drug. We used this information in the implementation section.
Questionnaire
Highlights
In order to confirm some of the problems mentioned by doctors and companies. We sent out a questionnaire. The results indicated a consistency for the need for cheaper drugs for breast cancer. The questionnaire also presented the public's lack of knowledge of breast cancer.
We also sent out questionnaires investigating the opinions on current targeted drugs; our audience included the general public, breast cancer patients and their relatives.
In our survey, we wish to inquire about people's opinions on the price of the drug, as this is very related to the public as well as patients.
From our discussion with the doctors, we came to an understanding that drugs for treating breast cancer are expensive. We wanted to see how other people, people who live in a society (unlike us students) think about it. So one of the questions was about the affordability of breast cancer drugs. The results were reasonable: more than 70% of the patients’ relatives believe that the price is quite expensive that some households will be unable to pay the bill over a long time (Fig. 1), while more than 80% of the general public holds the same opinion (Fig. 2). So, it would be very understandable when 90% of our respondents said that they hope for a lower-priced drug (Figure 1.4)
The doctors we interviewed told us the current awareness of breast cancer were low, we still held questions about that, so also sent a questionnaire to gather information on the public’s understanding of breast cancer, including its precaution, transmission pathways, methods for breast examination etc.
Breast self-examination is probably the most convenient and efficient way for the diagnosis of breast cancer at an early stage, and with medical intervention, a high survival rate can be achieved. The examination itself is a rather simple task that includes rubbing the breast and checking for abnormal bumps, though from our results, more than 70 recent of the respondents do not know how to do the examination (Fig.4).
We have also asked our respondents to identify some of the common methods for breast examination for preventing cancer that are done in hospitals, such as X-ray examination and B-scan ultrasonography. The results show the B-scan is the most widely known examination method, while more than 36%of the people could not identify any examination method (Fig.5).
For the transmission of cancer, our respondents were asked whether they think breast cancer can be passed by breastfeeding (It obviously cannot!) However, the result showed that nearly a third of the people chose the wrong answer.
Summary:
A lack of awareness of the examinations that could prevent cancer development was shown. Besides, there are some misunderstandings about breast cancer that should have been clarified. It is to our greatest pity that breast cancer, the disease that is potentially related to every woman and man, was currently not well understood by most of our respondents. Thus, we decided to include the publication of breast cancer and its prevention as a major part of our project in our Public engagement section.
Overcoming the Issues Within Drug Development
We've categorized the pharmaceutical issues we learned from our interviewees into Price, Specificity, Side Effects, and Stability. Below, we present our proposed solutions for each issue to formulate our design, all of which came up with our discussion with the experts.
Using Aptamers for lower price and specificy
Price
Price
One of the main focuses of pharmaceutical companies is to lower the price of the drug. As this can be more appealing to the general public. However, even after over 30 years of development, the current price of antibody drugs remains high, as they are hard to produce or maintain batch to batch qualities.
For example, the current payment needed for the PD-1 in China is 30,000 yuan per year with health insurance, and the price for the Lapatinib and Trastuzumab is approximately 110,000 yuan for one period of remedy in China with health insurance. Which is not very affordable for most patients with this disease.
Reflecting on the problems of price, we went on to research about aptamers, an idea proposed by Dr. Qiu and mentioned in our discussion with Pharmaceutical Companies.
Aptamers are ligands that can bind to molecules, but instead of being composed of proteins (like antibodies), it is purely ssDNA. The weak interaction forces between the nucleotides allow them to fold into unique structures.Aptamers have many advantages over antibodies[2]:
| Characteristics | Aptamers | Antibodies |
|---|---|---|
| Immunogenicity | Low | High |
| Internalization | Fast | Low Efficient |
| Modification | Easily Modifiable | Hard to modify |
| Batch-to-batch variation | High Uniformity of Product | Low Uniformity of Product |
| Production & Development Cost | Low | High |
| Production & Development Time | Short | Long |
| Versatility | Wide Range of Targets | Targets with Immunogenicity |
We also consulted with GreatBay_SCIE 2020, as their project is also regarding aptamers, though of different use. They advised us that we shouldn't focus too much on SELEX selection, as they've has already done so last year. They think the potential of aptamer lies in its ability to be modified and utilized easily; they advised us that spending time on how to implement the aptamer, improving existing aptamers perhaps would be a better option for us.
Specificity
Specificity is important, it is the foundation of targeting drugs. One of the most needed features of an ideal drug, from our interviews with doctors, is high specificity. Because this not only means that it has reduced side effects but also high effectiveness. Maintaining high specificity is key in our design.
To improve specificity even further, with our knowledge of cancer, and thorough research, we happen to encounter literature to make use pH-sensitive aptamer[3], which we used in our design. This allowed our aptamer to gain higher affinity under low pH near tumor tissue, which indirectly increases the specificity of our drug.
A small interlude we encountered with aptamers...
We encountered an issue with aptamer synthesizing, as pH-sensitive aptamer has extra length, many of which cannot be synthesized or too expensive to synthesize (synthesizing an ssDNA strand of 113 require 2000 RMB which is roughly 310 USD), so we sought help from our advisor Dr. Zong, pointed out there are other ways to synthesize them ourselves such as using asymmetric PCR[4], which we tried and succeeded in producing ssDNA strands ourselves (See in More in Lab Pages)
Using Nanoparticles for Less Side Effects and Better Stability
Side Effects
Side Effects
Chemotherapy although is the most mature type of drug treatment (developed in the 40s), it deals immense health damage to the patient.
Targeting drugs is the cornerstone to precision medicines. However, many current targeting drugs, especially ADC type, have issues regarding high toxicity. After talking with Dr. Qiu, we found out that there are currently, only two ADC drugs are approved by the FDA: TDM-1 and DS8201-a. Other drugs like MEDI4276 are all halted during the clinal phase due to high toxicity.
Intending to find a suitable payload of the drug that could lower side effects, we consulted Dr. Wei from the Northwest University of China, who is an expert in biomaterial and synthetic biology.
Dr. Wei suggests a nanoparticle drug delivery system, in which the drug was encapsulated inside a nanoparticle and the exterior side of the particle was conjugated with a factor that provides specificity for the targeted drug-aptamer.
We were informed that nanoparticles provide unparalleled advantages as a drug delivery system against tumors because of the EPR Effect (See more in Description & Proof of Concept) and its high customizability.
Dr. Wei points out that PLGA (poly lactic-co-glycolic acid) is a suitable material for the synthesis of our nanoparticle, for PLGA, first developed in the 1970s, is a polymer widely used in delivery systems, primarily because of its biocompatibility and degradability. It has been shown that the polymer can break down inside the body to produce non-toxic byproducts such as carbon dioxide and water[5]. In addition, Dr. Wei also suggests that as an alternative route, liposome, a lipid bilayer nano-sphere with similar properties to PLGA would also be suitable for our project[6]. So we decided to manufacture 2 types of modified nanoparticles: PLGA and Liposome.
We cooperated with Dr.Wei and Dr.Zeng, Dr. Shen to produce PLGA particles and Liposomes by using single emulsion-solvent evaporation and microfluidics technology respectively. (See more in Design and Proof Of Concept )
About Stability...
We raised this concern with Dr. Wei. Dr. Wei told us about factors such as PEG (polyethylene glycol) could be used to improve the plasma stability of our delivery system. PEG is a hydrophilic, flexible polymer that could be conjugated to the exterior of both PLGA nanoparticle and liposome[5]. And once conjugated to the surface of our system, the PEG layer forms a hydrophilic coating that avoids the opsonization of plasma protein; besides, due to the transient, rapidly changing structure of the polymer, the immune system would have difficulties in modeling an antibody around the system.
Using Doxorubicin...
Another problem we encountered was that nanoparticles were only a delivery system, we don't actually know which drug to use is most effective or suitable. Dr. Lu advised us to use Doxorubicin (DOX), which is used many times in innovative drugs and past literature; it can be considered to be a model drug for testing our cancer-curing methods.
Feedback
At last, we presented our design of using aptamers-conjugated nanoparticle drugs to previous experts to receive feedback.
We presented to the doctors and although all three doctors are not experts in drug design, they all think an aptamer-nanoparticle-based drug delivery system is promising in the future. One doctor argued that the need for drugs to treat HER2-positive breast cancer have always been there. Secondly, currently, there are no targeted drugs using aptamers on the market, so our project is innovative.
The pharmaceutical company told us that our project is the front research of developing new pharmaceutical methods with potential. By using aptamer as the guider, this variety of ADC drugs is still not on market, there is still, a gap to be filled.
The other pharmaceutical company also advised us how to prove our drug to be valid. Although limited by iGEM, we cannot go into any clinical phase, the company introduced to us in vitro methods to prove the validity of our drug. They say that our validity test must include results that the drug is effective against cancer cell lines. For our current progress, they just advised us to consider things like cytotoxicity assay, pharmacokinetics, using algorithms to simulate potential toxicity. Though we only chose to do cytotoxicity assay due to time, their advice was invaluable.
Dr. Qiu then instructed us on culturing cells. He advised several cell lines for us to use and guided us through steps of subculture and preservation.
We cultivated our cells at Shenzhen University and the students there helped us carry out our experiments.
Modeling
To aid our experiment better, we also developed models to help us predict our product as well as infer important information such as the amount of aptamers we should use to help achieve better results.
Structural Modeling
For the structural modeling part, in order to make our model of aptamer binding with protein visible, we have used various modeling tools(Matlab, RNA Composer, mfold) to help us get the individual model of the aptamer and the protein. But we found it difficult to bind the models of the aptamer and the protein together. To solve this problem, we collaborated with the team ShenZhen_SMS to learn how to use Discovery Studio to attach the protein and the aptamer together. In return, we provided suggestions about Blender which helped them visualize their protein better and also help them to put their 3D models onto the wiki.
Mathematical Modeling
For the mathematical part of the modeling, Max Chen, a member of Greatbay_SCIE last year has provided valuable ideas of modeling, for example, he has mentioned that the distance between the aptamer should be greater than the diameter of an HER-II molecule so that the aptamer would not block the HER-II from binding onto it. The idea is further explored by our team by constructing the model of a liposome using the spherical coordinates and expressing the position of the aptamer using the coordinates, which allows us to analyze the optimum aptamer concentration mathematically, his idea has provided a valuable direction of our models.
To see more information on our modelling, click here to go to the modelling page.
Issues Remaining In Society
Lack of awareness
Despite the solutions we proposed earlier to overcome barriers in drug production, we realized that curing cancer is incredibly hard after talking and interviewing with doctors and specialists. It's is not an acute disease but rather a chronic one most of the time. Diseases like breast cancer have no vaccines, thus the best way to prevent this disease is to live a healthy life; or treat it as early as possible. However, the current state of public knowledge with breast cancer worries as, as seen from the questionnaire. Hence, we wish to make awareness about breast cancer. (Seen more in our Education & Public Engagement Page)
Reference
- Sung, H, Ferlay, J, Siegel, RL, Laversanne, M, Soerjomataram, I, Jemal, A, Bray, F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021: 71: 209- 249. https://doi.org/10.3322/caac.21660
- Hongguang, S., Jingsan, Z., Jianbo, W., Youli, Z. & Zhu, X. (2016) Aptamer Technology and Its Application In Tumor Diagnosis and Therapy. Prog Pharm Sci Aug. 2016 Vol. 40 No. 8.
- Thompson, I., Zheng, L., Eisenstein, M., & Soh, H. T. (2020). Rational design of aptamer switches with programmable pH response. Nature communications, 11(1), 2946. https://doi.org/10.1038/s41467-020-16808-2
- Marimuthu C, Thean-Hock Tang, Soo-Choon Tan, Chee-Hock Hoe, Rajan Saini, Junji Tominaga and Subash C.B. Gopinath songklanakarin J. Sci. Technol. 34 (2), 125-131, Mar. - Apr. 2012
- Parveen, S., & Sahoo, S. K. (2011). Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. European journal of pharmacology, 670(2-3), 372–383. https://doi.org/10.1016/j.ejphar.2011.09.023
- Lao, J., Madani, J., Puértolas, T., Alvarez, M., Hernández, A., Pazo-Cid, R., Artal, A., & Antón Torres, A. (2013). Liposomal Doxorubicin in the treatment of breast cancer patients: a review. Journal of drug delivery, 2013, 456409. https://doi.org/10.1155/2013/456409