The challenge Breast cancer is one of the most common cancers globally, threatening women's health. According to the World Health Organization (WHO) there were approximately 2.3 million new cases of breast cancer and 670,000 related deaths worldwide in 2022 alone[1]. Advancements in medical technology have introduced various treatment options for breast cancer, including chemotherapy, radiotherapy, and surgical interventions. Surgical treatment, often the most effective and commonly used option, involves the removal of tumor-affected breast tissue or the entire breast[2]. However, patients undergoing surgical treatment often face enduring psychological and physiological challenges. Follow-up examinations are central components of cancer survivorship care to prevent recurrence. Such examinations usually require patients to go to hospital from time to time, which costs a lot of time and medical resources. In addition, regular checkups in hospital places individuals in a state of "awaiting diagnosis," blocking their psychological transition from "patient" to "ordinary person”. Bergerot et al. (2024) pointed out, breast cancer survivors frequently exhibit significant emotional fluctuations before and after follow-up appointments, often accompanied by anxiety, panic, and sleep disturbances[3]. Therefore, there is a pressing need to improve the ways of follow-up examinations so that they could be convenient and less psychologically disturbing for breast cancer patients. Inspiration Recent years, breast reconstruction surgery has become an increasingly common choice for breast cancer patients after mastectomy. According to data released by the American Society of Plastic Surgeons (ASPS) in 2023, the number of breast reconstruction surgeries performed in the United States reached 157,740, marking a 4% increase[4]. The increasing number of patients undergone breast reconstruction surgeries led us to think about whether a diagnostic system could be integrated into breast reconstruction surgery. To explore this idea, we consulted Dr. Wan Wang and learnt that autologous adipocytes transplantation is already a well-established technique for breast reconstruction. Considered that autologous adipocytes transplantation is also widely applied in plastic surgery. we included women who undergone plastic surgery as one more potential target user of our project. From literature review, we learned that Adipocytes in tumor microenvironment are frequently induced to undergo transformation into cancer-associated adipocytes (CAA) through stimulation of cytokines secreted by cancer cells. We realized that the altered gene expression during transformation of CAAs could be ideal target for surveillance of cancer occurrence, as it reveals the existence of cancer secreted cytokines[5]. Another feature of adipocytes which makes them suitable as chassis cells for cancer surveillance is that, as terminally differentiated cells, adipocytes exhibit minimal proliferative capacity and limited motility, which ensures adipocytes’ long-term stability in vivo. Therefore, we came up with the idea to implant a cancer surveillance system during autologous adipocytes transplantation for women undergoing breast reconstruction surgery or plastic surgery, so that individuals might be able to monitor cancer occurrence without going to hospitals.
Working principle of ABCS
Adipocyte for Breast Cancer Surveillance, ABCS is a real-time cancer warning system specifically designed for women who have previously had breast cancer or undergone breast surgery. Procedure outline Adipocytes will be obtained from individual’s autologous adipose tissue. Through recombinant adeno-associated virus (rAAV) infection, plasmids with sensor elements to detect specific gene expression associated with CAAs will be delivered into these adipocytes. These engineered adipocytes will then be transplanted to breast tissue at breast reconstruction or plastic surgery, which sense the specific gene expression associated with CAA and secrete reporter molecules. Such surveillance system enables individuals to be aware of potential cancer activity.
Procedure outline of ABCS
Biomarker A key mechanism to transform adipocytes into CAA is the stimulation from cytokines that are secreted from cancer cells, which usually induce expression of their target genes in adipocytes. Therefore, we construct a sensor element in adipocytes, enabling them to detect such target genes. Through literature review and database analysis, we found that breast cancer cells secrete plasminogen activator inhibitor-1 (PAI-1) and C-X-C motif chemokine ligands (CXCLs), which upregulate the expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) and leukemia inhibitory factor (LIF) in adipocytes respectively. Based on these, we delivered a sensor element which can sense the upregulated expression of PLOD2 and LIF into adipocytes. In other words, PLOD2 and LIF are the target biomarkers for ABCS.
Interaction between cancer cell and CAA through cytokines
Sensor element——RADAR RNA sensing using adenosine deaminases acting on RNA (RADAR) is a novel RNA detection technology based on RNA editing, composed of three parts:
A marker gene for validation of successful infection (e.g. mCherry)
A sensor sequence that is reverse complementary to the target RNA
An output reporter gene
The core of the sensor is a central stop codon (UAG), which prevents the expression of the downstream reporter gene. When the trigger RNA is present, it binds to the sensor sequence, forming partial double-stranded RNA (dsRNA). However, due to the presence of a CCA sequence in the target RNA, an A-C mismatch occurs at UAG. This structural feature provides a substrate for ADAR enzymes, which bind and convert the adenosine (A) of the UAG stop codon into inosine (I), effectively releasing the inhibition of translation of the downstream output reporter gene. To enable simultaneous detection of 2 trigger RNAs, in our case, PLOD2 and LIF, we have designed a sensor element that connects two sensors in tandem to form an AND gate, ensuring that the reporter gene is only expressed when both PLOD2 and LIF are present[6].
Working principle of RADAR
Reporter genes Gaussia luciferase (Gluc), an enzyme that can catalyze with coelenterazine as substrate to generate luminescence, is our choice of downstream reporter gene. Gluc is nontoxic and naturally secreted, cleared mainly by the renal pathway, and can finally end up in urine[7-8]. Therefore, coelenterazine can be added into urine samples from patients implanted with ABCS and the present of luminescence in these samples indicated the occurrence of cancer cells. Future view In the future, we hope to offer ABCS as an alternative or complimentary choice of follow-up examinations for individuals that have undergone breast surgery. ABCS can function stably and safely over long periods, serving as a sentinel that reports cancer occurrence timely. We hope that this system could relieve breast cancer patients’ anxiety and burden of follow-up examinations in hospital.
Reference
1 Breast cancer 2024 March 13 https://www.who.int/news-room/fact-sheets/detail/breast-cancer.
2 NCCN breast cancer clinical practice guidelines 2024.
3 Bergerot, C., Philip, E. J., Bergerot, P. G., Alfano, C. M., & Ludolph, M. (2024).Is cancer back? Psychological issues faced by survivors of breast cancer. Frontiers in Psychology, 15, Article 1291054.
4 American Society of Plastic Surgeons (ASPS) 2023 Clinical Statistics Report.
5 Wu, Q., Li, B., Li, Z., Li, J., Sun, S., & Sun, S. (2019). Cancer-associated adipocytes: key players in breast cancer progression. Journal of Hematology & Oncology, 12(1), 95.
6 Kaseniit KE, Katz N, Kolber NS, Call CC, Wengier DL, Cody WB, Sattely ES, Gao XJ. Modular, programmable RNA sensing using ADAR editing in living cells. Nat Biotechnol. 2023 Apr; 41(4): 482-487.
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8 Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther. 2005 Mar; 11(3): 435-43.