This blog is dedicated to all of the individuals on this planet that passed on from the many types of brain cancers, and to the valiant practitioners that endeavor to treat their afflictions. This includes our daughter Amy. Amy’s brave mental, and physical strength along with her profound sense of humor, greatly attenuated our close-knit family’s suffering while she battled her dyer conditions. It our hope that the new concepts presented herein will be of clinical value to the physicians who are dedicated to solving the difficult problems that brain cancers present to their profession.
Surgery, radiation therapy, chemotherapy, steroid therapy and radio-surgery have been the mainstream management of brain cancers. Nonetheless, there are management changes associated with newer technological advancements. However, clinical problems still exist.
Some of the daunting difficulties and dyer side effects that brain cancer surgeons encounter are as follows:
Tumor recurrence after resection, Inability to Resect the Total Tumor, Micro-tumors Cells, Scar Formation, Loss of Function, Seizure Syndrome, Metatastic Lesions from Other Systemic Malignancies, and a Wide Range of Side Effect from Different Radiation Technologies along with Chemotherapeutic agents. It the intent of the concepts proposed herein that are based upon current research, to change the clinical approach and to treat a myriad of problems associated with currant brain cancer therapies.(1.) (2. )(3.) (4.)
The transplantation of autologous bone marrow derived mesenchymal stem cells (MCSs) is a vital part of the new suggested therapy. (5.) These cells would be extracted from the cancer patient a week or so prior to their surgeries. In a proper laboratory setting they would be induced to become neurons and glial cells. (6.)Without becoming involved in the morality or legal controversies, the cells that would induce the patient’s MCSs are the molecules obtained from the brain tissues of legally donated aborted fetuses that are at presently stored in at least 2 Fetal Cell Banks. One is in the UK (7.) The other is in Lausanne Switzerland. (8.) A prerequisite for the technologies presented would be a facility that would be capable of dealing with the extraction of the autologous MCSs with the capability to incubate them and have adequate microbological technologies in order to have them ready for the surgical procedure. The other important aspect of the therapeutic procedure will be a Kit whose stored lyophilized molecules that will be reconstituted at the time of the surgery. The Kit’s molecules are also relevant in the induction protocols. The detailed protocols will be presented latter in this paper.
TECHNOLOGIES AND PRODUCTS NEEDED TO ATTAIN CONCEPTUAL SUCCESS
Patient derived (Autologous) Mesenchymal stem cells from bone marrow. (9.) (10.)
Doxycycline 100mg. Given orally t.i.d. 3-5 days: 1-2-days before; during the day of surgery; and 1-2 days after surgery. (11.) (12.) (13.) (14.)
autologous patient’s blood serum. (15.)
Kit that stores the following for a single surgical procedure:
2 vials containing 2-5 mg sterile lyophilized fetal derived neuronal ECM molecules(16.)
1 vial 2mg sterile lyophilized fetal derived neural cellular fibronectin. (17.)
Sterile water and sterile saline for reconstituting lyophilized molecules and site wash.
Sterile 5 cm poly(D,L-lactic acid-co-glycolic acid) (PLGA) sponge or nanoparticles.(18.)
RATIONALE FOR UTILIZING THE COMPONENTS IN PRECEDING PARAGRAPH
MESENCYMAL STEM CELLS (MCSs) AUTOLOGOUS
MCSs are functionally defined by their capacity to self renew and their ability to differentiate into neurons, astrocytes, and oligodendrocytes in vitro when grown under appropriate conditions.(20.) After the MSCs are cultivated in a conditioned medium containing human neural stem cells they express markers for neuron, astrocytes and oligodendrocytes. These differentiated cells obtained from this in vitro technology exhibited electrophysiological properties of neurons including action potentials. Further, animal studies have shown that that when these cells are transplanted into mouse brain
a confirmation was attained that these cells retained the capacity to differentiate in biologically active neuronal and glial cells in vivo. (21.)
The concepts herein proposes that by utilizing the patient’s own MCSs cells in a specific microbiological cell culture protocol these cells can be induced to become neuronal and glial cells. The inducing molecules will be the fetal derived neuronal ECM molecules that would be part of a specific growth media. The capacity of the MCSs cell to renew and divide, and with the induction input of medium laden fetal neuronal ECM molecules will induce their genetic capability to differentiate into the neuronal phenotype cells in a short period of time. These will be the cells to replace the surgically removed tumor with the potential to restore some of the normal brain activity. After there is confirmation that the patient’s MCSs have attained the neuronal phenotype in the culture media a research protocol decision will establish whether to fast cryopreserve the MCSs for later usage, or to use that culture immediately for the surgical procedure.(22.) During the surgical procedure these cells will be taken out of the culture vessel and soaked thoroughly in the PLGA sponge for its transplantation into the area of tumor removal site.
The media that has shown efficient growth capability of MCSs is Delbecco’s Modified Eagle’s Medium/F12 (DMEM/F12). (23.) It will be augmented with the Fetal Derived Neuronal ECM molecules, and the patient’s own serum. (15.) There will in vitro research to determine how long it would take to induce the MCSs to attain the neuronal phenotype.
DOXYCYCLINE:AN ESSENTIAL THERAPEUTIC AGENT FOR THEIS CONCEPT
All cancer cells produce a vast variety metalloprotienase enzymes (MMP) that destroy their adjacent tissues microinvironment, and lead to their invasiveness. It further promotes the most devastating aspect of all cancers metastasis.(11.) (12) (13.) There is even some research evidence that the MMP are instrumental n breaking down the Blood Brain Barrier (BBB) and permits metastasis to invade into and out of the brain. (24.)
Doxycycline and other tetracylines have shown irrefutably that they attenuate or completely inhibit the devastating effects of MMPs. Therefore, since brain cancer cells have shown great invasive propensities and a great tendency for metastasis, it is absolutely essential that this thoroughly clinically tested agent be apart of the therapy.
AUTOLOGOUS SERUM FROM THE PATIENT
Human serum has been found to be efficient in supporting proliferation and differentiation of human mesenchymal stem cells in vitro and in vivo. (15.) The use of the patient’s own serum will pose no health risk and will further simplify the cell culture procedures. Its presence in the media may shorten the culture induction time. Its transplantation into the surgical site along with the other components may enhance the total healing potential.
FETAL DERIVED NEURONAL EXTRACELLULAR MATRX MOLECULES (ECM)
Recent research has shown that microenvironment of normal human cell’s extracellular matrix (ECM) is a dynamic action zone that functions to instruct cellular phenotype. (25.) These cells produce and secrete into their peripheral environment the “exact” ECM molecules for the maintenance of their “specific differentiated phenotype. Neurons and astrocytes also follow these parameters.
The therapeutic agents, the EMC’s derived from fetal neuronal tissue cell cultures are lyophilized and available in the Kit that can be stored in a freezer. These molecules become biologically active when reconstituted. They are the induction agents that will instruct the patient’s MCSs to become the neuronal phenotype. All normal cells produce and excrete the exact ECM molecules needed for their own “specific differentiated phenotype”. The extracellular matrix molecules (ECM) are different for each differentiated group. Nerve cells such as astrocytes and neurons also are different. Their specific complex microenvironments are the determining factors to maintain the individual differentiation phenotype.
FETAL DERIVED NEURONAL LYOPHILIZED CELLULAR FIBRONECTIN
Cellular Fibronectin is a spectacular evolutionary molecule that is directly involved in a myriad of profound biological activities. The discoverer of cellular fibronectin was Dr. Richard O. Hynes in 1974 (26.) At that time it was the largest protein ever discovered, and showed an amazing in vitro ability. In 1977 when its purified solution was incubated with tumor cells, the malignant phenotype became more normal-like. (27.) This capacity for the conversion of tumors to be more normal was repeated by Dr. Ken Yamada. in 1979.(28.)
As research progressed for this protein many other discoveries came to light. Cellular Fibronectin plays a major role in cell adhesion, cell shape, cell surface architecture, growth, migration, and differentiation, and it is important for processes such a wound healing and embryonic development. Its research led to the discovery of the “Integrins”,the major cell surface family of protein receptors that form the transmembrame link between ECM glycoproteins and the internal structures of cells. It is believed that its two distinct chains mediate cell-cell and cell-matrix interactions and communications. Of the 21 known integrins, cellular fibonectin binds (ligands) to 10, more than any other protein. (29.)
Cellular fibronectin’s highly conserved genome has give rise to a array of molecules that play important roles in modern day metazoans. The blood clotting molecules, plasma fibronectin. fibrin, and Factor VIII, are progenies of cellular fibronectin. Other well known molecules such as vitronectin, tension, and titin, contain extended modular sequences from the more ancient cellular fibronectin.
Cellular fibronectin has shown to inhibit tumor recurrence at surgical wound site in an in vivo research study at Evanston Hospital. (30.) This effect was seen in the liver, the thigh, and most importantly in the brain. Cellular fibronectin in a FDA/IRB approved clinical trial for periodontal disease at UCLA was found to convert chronic diseased periodontal tissue back to normalcy without side effects. (31.) All pathogenic invasive microorganisms have receptors for cellular fibronectins. A plethora of research papers show that these pathogens invade human bodies through the Cellular Fibronectin molecules on the ECM. (32.)(33.) Cellular fibronectin is highly adhesive. When added during surgery it would bind and cover fibronectin receptors on pathogenic organisms. They would thereby be rendered inactivated and be unable to cause their infectious invasiveness. These abilities makes this well researched protein a valuable addition to the clinical applications of brain cancer surgeries.
Three short reviews are available and can be accessed on this website. “Cellular Fibronectin’s Role in Extracellular Matrix”, “ Cellular Fibronectin Extensive History” “Cellular Fibronectin as an Anti-microbial”.
STERILE SALINE AND/OR STERILE WATER
These solutions are used to reconstitute the lyophilized ECM molecules and the lyophilized cellular fibonectin molecules. The use of the sterile saline will reconstitute each of these components at ratio of 1 ml per 1 mg. If the lyophilized ECM and the cellular fibronectins are lyophilized with physiological phosphate buffer, then the products will be reconstituted with the sterile water., also at the ratio of 1 ml to 1 mg.
Sterile saline is also a valuable clinical application after removing all of the tumor. A wash of the tumor areas with the saline followed by a thorough aspiration will remove many tumorus micro-cells left after the surgery. These micro-cells have the propensity to cause recurrence and also gain entry in the lymphatics and venus drainage and become potential metastatic cells.
STERILE 5 CM PLGA SPONGES OR NANOPARTICLES
Poly(DL-latched-co-Glycoside) commonly known as PLGA is a FDA approved polymer for human usage and has been used a number years for drug delivery to a number of parts of the body. It is biodegradable and bio-compatable and can be made in many different compartmentalized sizes and also can be constructed to biodegrade in mater of minutes to hours or days. Their versatility makes this product ideal to contain the MCSs for the surgical mission expressed herein.
The mission of this paper is not to disrespect any of the honorable, brilliant physicians, that have the daunting task of preforming brain surgery. The mission herein is to show a possible methodology that has research credibility that may enhance the results of their efforts. These protocols to use bio-compatable polmeric materials for the controlled delivery of therapeutic agents to the brain is not a new concept. This technology has been in existence for some time enhanced by the effort of the Saltzman Research Group at Yale University. Professor W. Mark Salesman’s innovative technologies and philosophic approach to his chosen profession will lead to better methods for treating brain tumors. (34.) Perhaps the technologies suggested by this paper would be of this group ‘s interest.
The clinical usage of many diverse types of therapeutic agents imbedded into a variety of PLGA polymer configurations have been shown great therapeutic potentials. (18.) (19.) Therefore, the therapeutic concepts proposed in this paper have shown many years of clinical effectiveness. Nonetheless, in brain cancers and the surgeries related to same, to date, has not lead to appreciable prolongation of life nor a substantial alleviation of the untoward side effects relating to the surgeries, the chemotherapeutic drugs, and the radiation therapies.
PROTOCOLS FOR BRAIN CANCER SURGERIES
The author of this paper has the greatest respect and admiration for the brain surgeons whose vast education, training and mastering of the skills needed to pursue their chosen profession. Surgical procedures and their individualized protocols are absolutely not in any way questioned in this document. The suggested technologies herein have been created to enhance the healing outcomes to the total procedures.
+Human bone marrow-derived stem cells, MCSs, can be extracted relatively simply from the patient that will undergo a brain cancer surgery procedure. They can be maintained and expanded in culture. The have the capacity to be induced to become neurons and glia in culture. There is many research papers that show the success of this phenomenon.(5.) (6.) Therefore, the initial step before he surgical procedure is to extract an adequate amount of MCSs from the patient and culture plated in a Delbecco’s (DMEM) Media. An adequate amount of the patient’s blood is taken to make the patient’s serum.
- From the Kit described above one of the Feta Bain Derived ECM is reconstituted and added immediately to the media.
- Also added to the culture is the patient’s own serum. (15.)
- The information from previous research that determines the time-lapse that it takes for the conversion of the MCSs cell to become the neuronal is now taken into account. If there is a prolonged time-lapse the MCSs cell can be frozen and reconstitutes at the time of the surgery with out any appreciable loss in biological activity.
- 1 – 2 days before the surgical procedure the patient is give orally t.i.d., 100 mg doxycycline. Doxycycline t.i.d. is also given the day of the surgery and 1-2 days after the surgery.
- Day of surgery. Take the MCSs out of the culture and soak these now neuronal phenotype cells into the Sterile PLGA sponge. When the surgical procedure are near completion i.e., less that 10 minutes from its finish, the fetal derived cellular fibronectin and the second fetal derived ECM are reconstituted.
- The surgical site is rinsed with sterile saline and aspirated in order to remove any loose microscopic cell that may have tumor forming tendencies.
- Flood the total surgical site with the cellular fibronectin solution. Save some.
- Thereafter, flood the entire site with the ECM solution.
- Place the PLGA sponge in which the MCSs had therein been incorporated.
- Add some cellular fibonectin over sponge site.
- Close the surgical site. and add more cellular fibronectin over all closure sites.
DISCUSSION AND CONCLUSIONS
Though the methodologies suggested herein will need further research, there already has
been substantial studies that demonstrate a former precedents for each of the technologies presented. The undertaking to bring this technology into fruition will certainly be a group effort and will take a facility that has all of the scientific laboratory skills in order to fulfill that total concept. However, since the present and prevailing therapeutic methodologies of treating the various malignant brain cancers has not shown that these lesions can be healed nor any prolongation of life, perhaps its time to reconsider a different approach to treat this devastating human condition.
The microbiological technologies suggested in this concept have been all done before. Culturing fetal derived tissue. (7.)(8.) Culturing MCSs. (5.) Inducing MCSs to become neuronal phenotypes. (6.) Purifying cellular fibronectin. (17.) Purifying total ECM. ( done by author, unpublished) Usage of human serum. (15.) Using PLGA sponges in a myriad of clinical applications. (18.) (19.) Though all of the above technologies have had scrutinized scientific precedents, the total of these technologies to date, have never been applied clinically. Perhaps this can, in the very near future, be done for brain cancers.
1. Results after treatment of craniopharyngiomas: further experiences with 73 patients since 1997.Hoffmann BM., et al., J Neurosurg. 2012 Feb: 116(2):373-84 Pub 2011 Sep 23
2. Brain tumor epilepsy: a reappraisal and six remaining issues to be debated. Vermeil L. Rev Neuro; (Paris) 2011 Oct; 1677 (10 ):751-61. Pub 2011 Sep 3.
3. Surgical mortality at 30 days and complications leading to re-craniotomy in 2630 consecutive craniotomies for intracranial tumors. Lassen B. et al., Neurosurgery. 2011 May; 68 (5) : 1259-68; discussion 1268-69
4. Brain metastases: a medical neuro-oncology perspective. Chamberlain MC. Expert Rev Neurother 2010. Apr; 10(4): 563-73
5. Human mesenchymal stem cells cultures for neurol transplantation. Gordon D. and Scolding NJ. Method Mol Boil. 2009; 549: 103-118
6. Induction of neurol-like differentiation in human mesenchymal stem cells derived from bone marrow, fat, spleen and thymus Krampera M, et al., Bone 2007 Feb; 40 (2); 328-90
7. The Fetal Tissue Bank: Contact: Human Development Biology Resource (HDBR) Email: HDBR@ich.ucl.ac.ul
8. Consistency and safety of cell banks for research and clinical use: preliminary analysis of fetal skin cells. Quintin A, et al., Cell Transplant 2007: 16 (7) 675-84 Dr. Silke Mark Email: email@example.com
9. Mesenchymal stem cell differentiation and roles in regenerative medicine. Hwang NS., et al. , Wiley Interdiscip Rev Syst Boil Med. 2009 Jul-Aug; 1 (1) 97-108
10. Adult stem cells applied to tissue engineering and regenerative medicine. Cuenca-Lopez MD, et al., Cell Mol Boil (Noisy-regrind) 2008 Oct26; 54 (1): 40-51
11. Doxycycline use in patients with lymphangioleiomyomatosis: safety and efficiency in metalloproteinase blockade. Pimento SP, et al., J Bras Pneumol.2011 jul-Aug; 37(4); 424-30
12. Anti-invasion and anti-tumor growth effect of doxycycline treatment for human oral squamous-cell carcinoma–in vitro and in vivo studies. Shen LC, et al., Oral Oncol. 2010 Mar; 46 (3): 178-84 Pub 2009 Dec 29 9
￼￼13. Minicycline reduces glioma expansion and invasion by attenuating microbial MT1- MMP expression. Markovic DS, et al., Brain Behave Immune. 2011 May;25 (4) 624-8 Pub 2011 Feb 13
14. Extra matrix mettaloprteinase inducer is a negative prognostic factor of pediatric meduulloblastoma. Chu T, et al., Pathol Oncol Res. 2011 Sep 17 (3):705-11 Epub 2011 May 31
15. Human serum is as efficient as fetal bovine serum in supporting proliferation and differentiation of human multipotent stromal (mesenchymal) stem cell in vitro and in vivo. Aldahmash A, et al., Stem Cell Rev. 20011 Nov; 7 (4) 860-8
16. An efficient method for the culturing and generation on neurons and astrocytes from second trimester human central nervous system tissues. Barami K, et al., Neurol Res. 2001 Jun; 23 (4) 321-6
17. Binding of soluble form of fibroblast surface protein, fibronectin to collagen. Engvall E and Ruoslahti E. Int J Cancer 1977 Jul 15; 20(1): 1-5
18. PLGA Nanopartcles for ultrasound-Mediated Gene Delivery to Solid Tumors. Marxa Figured and Rinat Esenaliev. Journal of Drug Delivery vol. 2012 Article ID 767839, 20 pages doi; 10, 1155/2012/767839
19. Poly(epsilon-capropactone) and poly(L-lactic-co-glycol acid) ( PLGA) degradable sponges attenuate astrocyte response and lesion growth in acute traumatic brain injuries. Wong DY, et al., Tissue nEg. 2007 Oct; 13(10): 2515-23
20. Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells. MA K, et al., Neurol Res. 2011; 33(10): 1083-93
21. Intralesional mesenchymal stromal cell transplant in a rodent model of cortical cryoinjury. Moorthy RK, et al., Neurol India 2011 Jul-Aug;59 (4) 573-8
22. Evaluation of Bone Marrow-Derived Mesenchymal Stem Cells After Cryopreseration and Hypothermic Storage in Clinically Safe Medium. Ginis I, et al., Tissue nEg Part C Methods. 2012 Feb 2. (Epub ahead of print)
23. Effect of culture media on expansion properties of human umbilical cord matrix- derived mesenchymal cells. Salehinejad P, et al., Cytotherapy 2012 May 15 (Pub ahead of print)10
24. Matrix metalloprotienase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins. Feng S, et al., PLoS One 2011; 6 (8): e20599. Pub 2011Aug 17
25. Extracellular matrix remodeling and cellular differentiation. Streuli C, Current Opinion in Cell Biology 1999; 11: 634-40
26. Characterization of the external proteins of hamster fibroblasts. Hynes RO and Humphryes KC, J Cell Boil; 1974: Aug; 62 (2) : 438-48
27. Restoration of norma morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein. Ali IU, Manta V, Lanza R Hynes RO Cell 1977 May; 11 (1) : 115-26
28. Fibroblastic Cellular and Plasma Fibronectins are similar but not identical. Yamada KM and Kennedy WG,The Journal of Cell Biology Vol. 80 1979 pp.492-498
29. Integrins and signal transaction pathways: the road taken. Clark EA and Brugge JS, Science 1995 Apr 14; 268(5208) 233-9
30. The role of fibronectin in tumor implantation at surgical sites. Murphy MS, Scanlon EF Silverman RH, Goodheart CR, Goldschmidt RA, and Jelachich ML, Clin. xEp. Metastasis. 1993; 11, 159-173
31. The Effect of Fibronectin and a Bone Xenograft on Regenerative Treatment: A Feasibility Study. Camarco PM, et al., Compendium October 2006; 27(10) 560-69
32. Identification and characterization of a fibronectin-binding protein from Clostridium difficult. Hennequin C, et al., Microbiology Oct; 149(Pt 10): 2779-87
33. Staphylococcus aureus host cell invasion and virulence in sepsis is facilitated by the multiple repeats within FnBPA. Edwards AM,et al., PloS Pathog. 2010 Jun 24;6(6) e1000964
34. New Methods for Direct Delivery of Chemotherapy for Treating Brain Tumors. Sawyer AJ, et al.,Yale Journal of Biology and Medicine 79 (2006),pp. 141-152