Functions of Exosomes

Exosomes belong to extracellular vesicles as nanoparticles. They are intraluminal vesicles of multivesicular bodies. Exosome composition reflects its origin and its specific signaling mechanism. Proteins are essential components for exosomes and differ from one to another. For example.human monocytes released exosomes showed positive results for CD9, CD63, CD81, Tsg101 and Hsp70 flow cytometry and Western blotting [1].Other protein components such as receptors, transcription factors, enzymes, extracellular matrix proteins, lipids, nucleic acids (DNA, mRNA, and miRNA) can be inside and on the surface of the exosomes[2]. Lipid component of exosomes varies from lyosbisphosphatidic acid (LBPA), Sphingomyelin, phosphatidylcholine and BMP ( Bone morphogenetic proteins), yet sphingomyelin is the most common lipid component in exosomes[3,4]. Phosphatidic acid is a basic structure in previously mentioned lipid components. According to the tumor derived exosome vital existence within the tumor microenvironment, it has a major function in tumor initiation, progression, survival of cancer stem cells (CSCs),hypoxia induced epithelial mesenchymal transition, angiogenesis and chemotherapy resistance via cell-cell communication. It can transit signal to extracellular matrix cells such as fibroblasts, iimmune cells and endothelial cells by expressed proteins on its surface or inside it like miRNA, mRNA, incRNA[5,6,7].

Classification of Exosomes

There are two main categories of exsomes, the first category is tumor-derived exosomes (TDE) in which tumor cells secrete their own exosomes that show their own specific antigens and proteins and aid in tumorogenesis and spread as mentioned before. Thesecond one is normal cellular derived exosomes such as human umbilical vein endothelial cells, mesenchymal stem cells (MSC), T cells, B cells, macrophages, dendritic cells (DC), natural killer (NK) cells [8,9]. For example, mesenchymal stem cells (MSCS) , macrophage derived exosomes and monocyte derived exosomes. Bioavailability of this category is significantly high as it can be present in all body fluids such as saliva, milk, blood and cerebrospinal fluid. So that, these types can be used as targeting therapies for neuronal, cardiovascular diseases besides different types of cancer especially the immune-cell derived exosomes. There is another newly-biomimetic exosome type can be used as a therapeutic drug carrier and it is better in comparison to other immune cell derived exosomes, because it acts as a chemoattractant for different immune cells. They are called immune cell derived exosome mimetic (IDEM)[10]. That`s why, it is best used in immunologically cold tumors. Its advantages over other immune cell derived exosomes are included in adequate loading and encapsulation, sufficient extraction yields[11,12], high recovery and purity, lower cost, and well- standardized protocol[13]. ThP-1 monocytic cell line is selected for IDEM production due to its increased proliferation and variable immunotherapy approaches [14].

Cold Tumors

Cold tumors lack T-cell infiltration characteristics, so they elicit weak response to different immunotherapy results [15]. However, they are resistant to different immunotherapeutic approaches due to its hypoxic nature and cold microenvironment, they can be turned to hot tumors by generation reactive oxygen species (ROS) and IDEM.. ROS are produced in result of water splitting reactions such as oxygen (O2) self-supplementing conjugated microporous polymer nanosheets[15] and DNA damage which increases histone H2AX. Cold tumors such as breast, ovary, prostate, pancreas, and brain (glioblastoma) are usually hypoxic and demand high glucose needs for aerobic glycolysis. Warburg effect of hypoxic tumor is necessary for maintenance the appropriate carcinogenic hypoxic and acidic microenvironment [16].

Warburg Effect in Cancer Cell Glucose Metabolism

Cancer cells prefer aerobic glycolysis other than mitochondrial oxidation even in normoxic environment in spite of low ATP molecules production in comparison to mitochondrial oxidation. In aerobic glycolysis, just 2 ATP molecules are produced and lactate is produced. Also, glycolysis is the source of nucleotides, lipids and amino acids formation [17]. Another advantage of aerobic glycolysis is creation acidic tumor environment which is suitable for tumor progression and metastasis. Many carcinogenic signals and genetic mutations overexpress enzymes that are responsible for aerobic glycolysis and other tumorigenic effects [18].

Enzymes of Aerobic Glycolysis

Glucose transporters such as GLUT1, GLUT3 and GLUT12 are present on the cytomembrane. They participate in glucose uptake, respiration and metabolism mainly. Also, they have augmentative carcinogenic mechanisms, so that they are overexpressed in many cancer types [18]. GLUT1 enzymes are overexpressed in poor differentiated cancers, positive lymph node metastasis,big size tumors, and bad overall survival and disease-free survival for more glucose uptake [19]. GLU3 enzymes are abundant in glioblastoma [20], ovarian cancer [21], gastric cancer [22, 23],

and non-small cell lung cancer [23] , due to its high glycolytic efficiency. GLUT12 is upregulated in rhabdomyosarcomas, oligodendrogliomas, oligoastrocytomas, astrocytomas, and breast and prostate cancer [24].

Cytoplasmic hexokinases are responsible for intracellular glucose phosphorylation, however they promote glycolysis when they are bound to the mitochondria near the mitochondrial ATP [25]. Phosphofructokinase (PFK) subtypes which phosphorylate fructose-6-phosphates and other fructose phosphate derivatives in aerobic glycolysis, show maximum activity in hypoxia, P53 deletion in cancer, in result more glycolysis flux [26]. Finally, Lactate dehydrogenase (LDH) that converts pyruvate into lactic acid, the final form of glycolysis [27]. As i mentioned before, all glycolysis enzymes participate in chemotherapy resistance and tumor progression by many different signaling pathways[18]. Pyruvate kinase M2 (PKM2) stimulates glycolysis and entering the glycolysis products into branching pathways such as pentose phosphate pathway [28]. PKM2 is the dominant one in cancer cells and associated with aerobic glycolysis unlike PKM1 which is associated with mitochondrial respiration [29].PKM2 has two different states with two different functions; the first is tetrameric state which is responsible for production of pyruvate by phosphoenolpyruvate (PEP). The tetrameric state of PKM2 has high affinity to phosphoenolpyruvate (PEP). While the dimeric form of PKM2 has non-glycolysis function, after its translocation into the nucleus. It acts as a transcriptional factor to promote transcription many cancer cell genes [30]. It binds with histone H3 and phosphorylates histone H3 at T11 upon EGF receptor activation [31]. The mitogenic and the oncological nuclear function of PKM2 necessitate acetylation at K433 to suppress its binding with fructose 1,6biphosphate (FBP), in other words, upregulatesnonmetabolic function of PKM2 and allows it nuclear accumulation for genetic transcriptional activity. [32,33]

I131beta Rays Emission, Its Clinical Applications and DNA Damage

Ionizing irradiation initiates many possible hazardous effects to the cell and DNA. I-131, a beta-emitting isotope (606 keV, 90%) and a half-life of 8.02 days [34], When the radioactive I¹³¹ containing molecule is translocated into the nucleus, it can damage the cell DNA, improper DNA replication or severe DNA damage and apoptosis. Another possible effect of DNA damage is generation reactive oxygen species by formation hydrogen peroxide H₂O_2. . However, the resulted DNA can cause initiate apoptosis, the malignant cell can save itself by many DNA repair mechanisms. [35-38] Radioactive iodine I¹³¹ is used for treatment thyroid carcinoma with little damage to surrounding tissues (more than 2.0 mm from the tissues absorbing the iodine). It is also used for treatment of neuroendocrine tumors, including neuroblastomas, paragangliomas, and pheochromocytomas [39].

Biotin as a Vitamin-Mediated Drug Targeting

Cancer cells have high replication rate and express certain essential vitamin receptors on their cell surfaces for vitamin internalization. So that, many vitamins have been of great interest for targeting chemotherapeutic drugs towards malignant cells. (tumor-specific vitamin-receptor mediated endocytosis) [40]. For example, biotin conjugates are used for targeting tumors such as biotin- SBT-1214 conjugate, the biotin-fluorescein conjugate, the biotin-coumarin conjugate, biotin-doxorubicin conjugate and biotin gemcitabine. Conjugation the previously mentioned drugs to biotin limits its toxic effects [41]. Biotin also is used for combination with p53 protein, then loaded into C2-streptavidin transporter. After then, C2-streptavidin transporter is introduced into certain mammalian cells by a clathrin-dependent endocytosis [42].

Summary of the Suggested Invented Molecule and Its Anti-Tumor Effect

Pyruvate kinase M2 is dominantly present in many cancer cell types, and it is considered one of the most essential enzymes for aerobic glycolysis in tumor cell metabolism. But, when its lysine residue 433 is acetylated, it will act as a transcriptional factor[32,33]. In addition, FGFR-1 dependent tyrosine phosphorylation will be suppressed. Tyrosine 105 phosphorylation will shift the function of PKM2 from a transcriptional factor to a metabolic function [43].

_.My aim of the modified PKM2 is the following:

• Acetylation of lysine 433 to be translocated into the cancer cellwith biotin molecule. Here, there will be 2 targeting molecules which are PKM2 itself and biotin. (figure. 1).
• Dephosphorylation of tyrosine105 to inhibit its cytoplasmic metabolic function (figure.1).
• Attachment of 3 molecules of radioactive iodine I ¹³¹ to the dephosphorylated tyrosine 105 residues (figure. 1).
• Encapsulation of the modified PKM2 into monocyte derived IDEM to yield the final molecule (figure.2)

Figure 1: Structure of Modified Pyruvate Kinase M2.

Figure 2: Modified PKM2 Encapsulation with monocyte derived IDEM.

I¹³ PKM2-IDEMexosome molecule will reach to malignant cell microenvironment then to tumor cells by three targeting substructures (fig.3A). The first is monocyte-derived IDEM exosome, the second is PKM2 itself which is predominantly present in malignant cell whose metabolism is aerobic and in bad need for PKM2 for Warburg effect, and the third is biotin molecule (vitamin- targeting mechanism). The molecule can affect tumor associated macrophages, cancer stem cells and the primary tumor cells (fig.3A). Cancer cells will uptake the biotin-acetylated PKM2 within its nucleus especially after tyrosine 105 dephosphorylation, as a transcriptional factor (fig.3B). Here, the role of the 3 radioactive iodine I¹³¹ molecules attached to the y105 comes by emission β rays. In a result, DNA damage will occur directly by beta rays and indirectly by releasing reactive oxygen species which will convert the cold tumor in a hot one (fig.3C). I can say that molecule is considered as a radiotherapuetic molecule (direct action) and an immunotherapeutic molecule (indirect action) by releasing ROS for attraction macrophages and different natural

Figure 3A: Diffusion of the therapeutic molecule from the circulation to the malignant cells within the tumore site, tumore-associated macrophages and cancer cells within the tumor microenvironment.

Figure (3B): Tramslocation of biotin acetylated PMK2 carrying radioactive iodine (I131) into the nucleus a transcriptional factor.

Figure (3C): DNA fragmentation by beta rays emission of I131 radioactive iodine.

Materials and Methods for Manufacturing IDEM-I¹³¹ PKM2

• Purification of dephosphorylated biotin-acetylated PKM2: rat lung cells were approved for 840 fold purification and overall yield 20%. The enzyme showed a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of Mr = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M2. Optimum enzyme PH is 6.5[44]. Glucose 5.5 mM is added to the culture without addition fructose 1,6 biphosphate. Instead Biotin will be added besides p300 acetyltransferase for K433 acetylation with biotin for promotion its nuclear translocation instead of K433 binding with its allosteric activator fructose 1,6biphosphate. [33, 45]The final product of the purification is K433-biotin PKM2.
• I ¹³¹ iodination ofdephosphorylated biotin-acetylated PKM2: Oxidation with sodium periodate in solid phase extraction of N-linkedglycopeptides. The oxidation of BSA and PKM2 was done following the sample preparations steps according to the extraction of N-linked glycopeptides. Briefly, 1 mg of BSA was reduced with DTT (5 mM final concentration) for 1 h at 60 °C and then alkylated with iodoacetamide (12 mM final concentration) at room temperature for 30 min in the dark. PKM2 does not contain any cysteine residues (not oxidized or reduced). Both proteins aredigested with trypsin at a 1:50 enzyme-to-protein ratio, overnight at 37 °C, with gentle rotation. Samples were desalted by solid phase extraction (SPE) HLB cartridges according to the manufacturer’s instructions. The peptides were collected in 1.5 mL of 50% acetonitrile in 0.1% TFA. A time-course oxidation was performed by adding sodium periodateI 131(30 mM final concentration in the dark at room temperature) to BSA and PKM2 digests for 15, 30, 45, and 60 min in separate reaction tubes. An oxidation control (no sodium periodate) was run simultaneously and labeled as time 0 min for plotting purposes. The oxidation at each time point was stopped by a 10-fold dilution with water and immediately followed by a SPE clean-up step. Samples are finally dried with a SpeedVac (20 °C) and reconstituted in 20 μL of a water:acetonitrile:formic acid (95:5:0.1) solution containing 40 fmol/μL Glu1-fibrinopeptide B used as an internal standard. Following steps will be continued during SPEG (Solid-phase extraction of N-linked glycopeptides) up to purification the tri-iodinated I131 tyrosine 105 of dephosphorylated biotin-acetylated PKM2 [46,47].
• 3- Purification of monocyte TP-1 cell line derived IDEM and encapsulation of tri-iodinated I 131biotin-PKM2[10]: Monocytic cell line (ThP-1 cells) was purchased from ATCC. Cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (ATCC 30-2001TM) containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin.ThP-1 were maintained at a concentration of 1-5 * 105cells/ml for expansion. Ovarian cancer cells (SKOV-3 cells) were purchased from SigmaAldrichand cultured McCoy’s 5A media(Gibco) containing 10% (v/v) fetal bovine serum, 100 U/mLpenicillin, 0.1 mg/mL streptomycin. Culture conditions were established at 370C and 5% CO2. Immune derived exosomemimetics were synthesized by optimizing the procedure, where cells are passed through porous membranes of decreasing size in order to be deconstructed and reconstructed consequentially. Briefly, ThP-1 cells (8.5 *106) are harvested and washed twice in PBS. The PBS-resuspended pellet is then filteredthrough 10 mm-filter PierceTM spin cups and centrifuged at 14,000 * g for 10 min at 40C. The pelleted flow-through is resuspended in PBS and the same process repeated. Consequently, the pellet is passed through 8 mmfilters with the same centrifuge settings as before. The pellet is finally resuspended in 150 µL of 0.22 µm-filteredPBS and run through G-50 Sephadex high capacity spin columns for further purification of thesolution. This solution was stored at -800C or used fordownstream applications.ThP-1 cells (8.5 * 106) are incubated overnight in 15mlRPMI-1640 media supplemented with Exo-Free FBS.Media is then collected and processed through a series of centrifugations to remove the cellular component (500 * g for 5 min) and any debris (2000 * g for 30 min). The remainingsupernatant is passed through 0.22 µm PES membrane filterand then concentrated using 10 KDaAmiconultracentrifugal filters. Total exosome isolation reagent (TEIR, Invitrogen) is then added in a 1:1 ratio to the volumeobtained after the Amicon-based concentration process. Thesolution is mixed by vortexing for 30 s and incubated overnight at 40C. The next day, the sample was centrifuge at 10,000 * gfor 1 h at 40C. The concentrated solution is centrifuged at10,000 *g for 1 h at 40C, and the pellet was resuspended in 0.22 mm filtered PBS. This solution was stored at -800C or used for downstream applications. Loading tri-iodinated tyrosine biotin-PMK2 by saponin as an encapsulation enhancer. A 0.1% concentration of saponin was tested. Briefly, both nanoparticlesolutions are mixed with 400 mg/mL of I131 PMK2 before addingsaponin, and the mixture is incubated for 5 min at370C in agitation at 200 rpm. Consequently, unencapsulatedI131-PKM2 removed using an Exosome Spin Column(Invitrogen). After the loading, the encapsulation efficiency(EE%) is measured. To this, 0.1% of Triton-X-100 was added to the samples for 10 min at RT. The concentration ofI131 PKM2 encapsulated in both IDEM (IDEM-I131 PKM2) andEXO (EXO-I131 PKM2) is determined by measuring its excitationand emission values (480 and 610 nm, respectively) againsta set of known standards with a Synergy H4 Hybrid PlateReader.
• 3-Assessment Of I131-Pkm2 Activity In Cold Tumor Cancer Cell Lines (Breast: Hs578t, Mcf-7; Prostate Pc3, Lncap): Cell viability is analyzed using an MTT assay, while cell apoptosis and cell cycle arrest are determined using flow cytometry. Reverse transcription?quantitative poly­merase chain reaction (RT?qPCR) and western blot analyses are performed to determine the changes in the expression levels of p53, B?cell lymphoma 2 (Bcl?2), Fas and growth arrest and DNA damage?inducible 45 (GADD45), following I131-PKM2 treatment [48].Also, reactive oxygen species can detected by flow cytometryusing H2DCFDA (cell-permeable fluorogenic probe compatible with phenol red, FBS and BSA).

In my innovated medication, usage of biotin and PKM2 as targeting molecules is a gold standard for cold tumors treatment. Modified PMK2 has nonmetabolic mutagenic function especially after translocation to the nucleus. Radioactive iodine will have dual therapeutic actions, its direct beta emissions on DNA molecules and reactive oxygen species generation. So, IDEM- I 131biotin PMK2 can be used as a first line therapy prior to any monoclonal antibody or chemotherapy. We can achieve maximum benefit for cancer patients with the least possible side effects.

1.        Ekstrom K, Omar O, Graneli C, Wang X, FrrughVazirisani, Peter Thomson. Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells; PLoS One. 2013; 8: 75227.

2.        Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular organelles important in intercellular communication. J Proteome. 2010; 73:1907-1920.

3.        Huotari J, Helenius A. Endosome maturation. EMBO J. 2011; 30:3481-3500.

4.        Taylor DD, Akyol S, Gercel-Taylor C. Pregnancy-associated exosomes and their modulation of T cell signaling. J Immunol. 2006; 176:1534-1542.

5.        Miyoshi H. Wnt5a potentiates TGF-β signaling to promote colonic crypt regeneration after tissue injury. Science. 2012; 338:108-13.

6.        Neumüller RA, Knoblich JA. Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer. Genes Dev. 2009; 23:2675-99.

7.        Deep G, Panigrahi GK. Hypoxia-induced signaling promotes prostate cancer progression: exosomes role as messenger of hypoxic response in tumor microenvironment. Crit Rev Oncog. 2015; 20:5-6.

8. Cheng L, Zhang K, Wu SY, Cui MH, Xu TM. Focus on mesenchymal stem cell-derived exosomes: opportunities and challenges in cell-free therapy. Stem Cells Int. 2017; 2017:1-10.
9. Zhao DY, Yu ZC, Li Y, Wang Y, Li QF, Han D. GelMA combined with sustained release of HUVECs derived exosomes for promoting cutaneous wound healing and facilitating skin regeneration. J MolHistol. 2020; 51:251-2263.
10. Pisano S, Pierini I, Gu J, Gazze A, Francis LW, Gonzalez D, Conlan RS and Corradetti B Immune (Cell) Derived ExosomeMimetics (IDEM) as a Treatment for Ovarian Cancer. Front. Cell Dev. Biol. 2020; 8:553576.
11. Ha D, Yang N, adithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm. Sin. B 6, 287-296. 2016.
12. Akuma P, Okagu OD, Udenigwe C.C. Naturally occurring exosome vesicles as potential delivery vehicle for bioactive compounds. Front. Sustain. Food Syst. 2019; 3:23.
13. Li X, Corbett AL, Taatizadeh E, Tasnim N, Little JP, Garnis C, et al. Challenges and opportunities in exosome research-Perspectives from biology, engineering, and cancer therapy. APL Bioeng. 2019; 3:011503.
14. Corradetti B. The Immune Response to Implanted Materials and Devices: The Impact of the Immune System on the Success of an Implant. Berlin: Springer International Publishing, Cold Tumors: A Therapeutic Challenge for Immunotherapy Paola Bonaventura.
15. TalaShekarian, Alcazer V, Valladeau-Guilemond J, Valsesia-Wittmann S, Amigorena S, Christophe Caux and StéphaneDepil. Front. Immunol. 2019; 10.
16. Reversing Immunosuppression in Hypoxic and Immune-Cold Tumors with Ultrathin Oxygen Self-Supplementing Polymer Nanosheets under Near Infrared Light Irradiation. Wei Jiang, Lei Wang, Qin Wang, Han Zhou, Yinchu Ma, Wang Dong, HangxunXu, Yucai Wang, advanced functional materials/ volume 31/ 2100354.
17. Warburg O. The Metabolism of Tumors. In: Constable & Co Ltd 1930.
18. Liu C, Jin Y, Fan Z. the Mechanism of Warburg Effect-Induced Chemoresistance in Cancer. Front. Oncol. 2021; 11:698023.
19. Barron CC, Bilan PJ, Tsakiridis T, Tsiani E. Facilitative Glucose Transporters: Implications for Cancer Detection, Prognosis and Treatment. Metabolism 2016; 65:124-139.
20. Libby CJ, Gc S, Benavides GA, Fisher JL, Williford SE, Zhang S, et al. A Role for GLUT3 in Glioblastoma Cell Invasion That is Not Recapitulated by GLUT1. Cell AdhMigr. 2021; 15:101-15.
21. Ancey PB, Contat C, Meylan E. Glucose Transporters in Cancer - From Tumor Cells to the Tumor Microenvironment. FEBS J. 2018; 285:2926-2943.
22. He Z, Chen D, Wu J, Sui C, Deng X, Zhang P, et al. Yes Associated Protein 1 Promotes Resistance to 5-Fluorouracil in Gastric Cancer by Regulating GLUT3-Dependent Glycometabolism Reprogramming of TumorAssociate Macrophages. Arch BiochemBiophys. 2021; 702:108838.
23. Ali A, Levantini E, Fhu CW, Teo JT, Clohessy JG, Goggi JL, et al. CAV1 - GLUT3 Signaling is Important for Cellular Energy and can be Targeted by Atorvastatin in Non-Small Cell Lung Cancer. Theranostics 2019; 9:6157-74.
24. White MA, Tsouko E, Lin CC, Rajapakshe K, Spencer JM, Wilkenfeld SR, et al. GLUT12 Promotes Prostate Cancer Cell Growth and is Regulated by Androgens and CaMKK2 Signaling. Endocr-Relat Cancer. 2018; 25:45369.
25. Bock FJ, Tait SWG. Mitochondria as Multifaceted Regulators of Cell Death. Nat Rev Mol Cell Biol. 2020; 21:85-100.
26. Imbert-Fernandez Y, Clem BF, O’Neal J, Kerr DA, Spaulding R, Lanceta L. Estradiol Stimulates Glucose Metabolism via 6-Phosphofructo-2- Kinase (PFKFB3). J BiolChem. 2014; 289: 9440-9448.
27. Van Wilpe S, Koornstra R, Den Brok M, De Groot JW, Blank C, De Vries J, et al. Lactate Dehydrogenase: A Marker of Diminished Antitumor Immunity. Oncoimmunology.2020; 9:1731942.
28. Fukuda S, Miyata H, Miyazaki Y, Makino T, Takahashi T, Kurokawa Y, et al. Pyruvate Kinase M2 Modulates Esophageal Squamous Cell Carcinoma Chemotherapy Response by Regulating the Pentose Phosphate Pathway. Ann SurgOncol . 2015; l 3:1461-1468.
29. Bluemlein K. No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget. 2011; 2: 393-400.
30. Wu S, Le H. Dual roles of PKM2 in cancer metabolism. ActaBiochimBiophys Sin. 2013; 45:27-35.
31. Yang W, Xia Y, Hawke D, Li X, Liang J, Xing D, Aldape K, Hunter T, Alfred Yung WK, Lu Z. PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis. 2012; 150:685-696.
32. SIRT6 deacetylates PKM2 to suppress its nuclear localization and oncogenic functions. Bhardwaj A, Das S. Proc. Natl. Acad. Sci. U.S.A. 2016; 113:538-547.
33. Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization Lv L, Xu YP, Zhao D, Li F L, Wang W, Sasaki N, Jiang Y, Zhou X, Li TT, Guan KL, Lei QY, Xiong Y. Mol. Cell. 2013; 52:340-352.
34. Audi G, Bersillon O, Blachot J, Wapstra AH. The NUBASE evaluation of nuclear and decay properties. Nucl. Phys. A 2003, 729, 3-128.
35. Advisory Committee on Human Radioactive Experiments. (n.d.). How does radiation affect humans?Georgetown University.
36. Canadian Nuclear Safety Commission. Types and sources of radiation. 2015.
37. National Human Genome Research Institute. Deoxyribonucleic acid (DNA) fact sheet.
38. Non-Destructive Testing Resource Center. (n.d.). Cell radiosensitivity.
39. Kumar K, Ghosh A. Radiochemistry, Production Processes, Labeling Methods, and a ImmunoPET Imaging Pharmaceuticals of Iodine-124.Molecules 2021; 26: 414.
40. Chen S, Zhao X, Chen J, Chen J, Kuznetsova L, et al. Mechanism-based tumor-targeting drug delivery system. Validation of efficient vitamin receptormediated endocytosis and drug release. BioconjugChem . 2010; 21: 979-987.
41. Tripodo G, Mandracchia D, Collina S, Rui M, Rossi D New Perspectives in Cancer Therapy: The Biotin-Antitumor Molecule Conjugates. Med chem. 2014; 1: 004.
42. Fahrer J, Schweitzer B, Fiedler K, Langer T, Gierschik P, et al. C2- Streptavidin Mediates the Delivery of Biotin-Conjugated Tumor Suppressor Protein p53 into Tumor Cells. BioconjugateChem. 2013; 24: 595-603.
43. TRIM35 Interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinomaOncogene2015; 34: 3946-56. 
44. Purification and properties of pyruvate kinase type M2 from rat lung panel Beate Schering , Erich Eigenbrodt , Dietmar Linder , Wilhelm Schoner Biochimica et Biophysica Acta (BBA) - General Subjects. 1982; 717: 2337-347.
45. JMJD8 Regulates Angiogenic Sprouting and Cellular Metabolism by Interacting With Pyruvate Kinase M2 in Endothelial Cells Jes-NielsBoeckel, AnjaDerlet , Simone F Glaser , Annika Luczak , Tina Lucas , Andreas W Heumüller , Marcus Kruger , Christoph M Zehendner , David Kaluza , AnuradhaDoddaballapur , KishoOhtani , KarineTreguer , Stefanie Dimmeler, ArteriosclerThrombV Biol. 2016 ;36:1425-1433.
46. Alejandro M. Cohen, RipsikKostyleva, Kenneth A. Chisholm, Devanand M. Pinto; Iodination on Tyrosine Residues During Oxidation with Sodium Periodate in Solid Phase Extraction of N-linked Glycopeptides, J. Am. Soc. Mass Spectrom. 2012;23:68-75,
47. Tian Y, Zhou Y, Elliott S, Aebersold R, Zhang, H. Solid-phase extraction of n-linked glycopeptides. Nat. Protoc.2, 334-339. 2017.
48. Weixiao Zhang, Rui Gao, Yan Yu, Kun Guo, Peng Hou, Mingqi Yu, Yan Liu And Aimin Yang, Iodine-131 Induces Apoptosis In Htori-3 Human Thyrocyte Cell Line And G2/M Phase Arrest In A P53-Independent Pathway, Molecular Medicine Reports.2015; 3148-3154, 2015,