Document:

ex10-13.htm

Exhibit 10.13

EXCLUSTVE LICENSING / MARKETING AGREEMENT

This Licensing Agreement ("Agreement") is made and entered into this 20th day of March, 2012, by and between GLIE LLC DBA , True2Life, a California Limited Liability Company, (referred to herein as "Licensor"'), and AL International, Inc., a Delaware Corporation, DBA Youngevity and DBA DrinkACT.com (refe1Ted to herein as '"Licensee").

Whereas Licensor is an established corporation in the marketing and sale of products related to dietary supplement products and has developed a distributor organization of independent authorized agents for the sale of its products, including the True Boost and other "True·• product brands. Whereas Licensee wishes to license the rights to sell Licensor's products and use Licensor's distributor organization and Licensor wishes to sell and/or transfer, among other things, its distributor organization and the True2Life product line(s) and this Agreement is to witness the following:

WITNESSETH:

    1.  Exclusive License of Business Assets.  Licensor shall grant an exclusive license to the Licensee, free from all liabilities and encumbrances except as hereinafter set forth, subject to the terms and conditions set forth in this Agreement, the following described property: Exclusive, worldwide rights to all business assets and properties owned or utilized by Licensor used in its regular coarse of business including but not limited to: Licensor's distributor organization, rights, intellectual property (including websites, and URLs), trademarks, and tradenames associated with the True2Life brand(s) and product line(s), Any product inventory (product inventory not purchased, will be held on consignment) , as well as other assets and rights to be able to carry on the business as if Licensor was still running the business ("Licensed Property").

    2.  License Fees. Licensor shall receive from Licensee a fee for the exclusive use of the Licensed Properly paid in the following ways:

       A.)  Royalty Fee: Licensor shall receive ten percent (10%) Royalty Fee, of the Net Sales from all members of Licensor's distributor organization (do\vnline distributor sales) for the first forty-eight (48) months.  4% will be calculated and paid for an additional 36 months (a total of 84 License Payments).   Net Sales are sales made by the distributor network (no matter which products are purchased) , not including taxes of any kind and shipping and handling, and less any refunds.

       Example:

       Months 1-48 are to be calculated at 10% 

       Months 49-84 are to be calculated at 4%

       B.)  Product Royalty: Licensor shall receive five percent (5%) Royalty Fee, of  Net True 2 Life Products sales outside of the True 2 Life Distributor Network, that is to anyone that is not now or in the future in the True 2 Life Distributor Group. This Royalty will be paid for 48 months, 2% for the next 36 months (a total of 84 Royalty Payments) in additional to the 10% Royalty Fee calculated and paid of the True2Life Distributor Organization  sales.

       Example:

       Months 1-48 are to be t:alculated at 5% 

       Months 49-84 are to be calculated at 2%

       C.)  Bonus Payment. If, within twenty-four (24) months after closing, sales of the Licensed Distributor Network are $50,000 or greater for 3 consecutive months, Licensee shall receive a bonus payment of $10,000.  If, within forty-eight (48) months after closing, sales of the Licensed Distributor Network are $150,000 or greater for 3 consecutive months, Licensee shall receive an additional bonus payment of $10,000. A total extra bonus potential of $20,000.

 

  

-1-

  

 

       D.)  Commissions in Genealogy. Dr. Luis and Evelia Arriaza shall be incorporated into the genealogy system for determining commissions within Licensee's systems just  above the Aniaza distributor network that is being leased as part of this transaction.  This will enable Dr. Luis and Evelia Arriaza to receive regular commissions on the sales of distributors in their downlines.  Commissions will be calculated using Licensee's regular commission structure and at regular intervolves.

       E.) Buy out. At the end of the Royalty Fee period , described above, Licensee shall have the right to purchase the Licensed Property for One dollar ($1.00). Ifat the end of the Royalty Fee period, Licensee chooses not to acquire the Licensed Property, all new distributors that enlisted during the period would be Licensees.

    3.  Payment of Purchase Price. The purchase price, as provided in Paragraph 2, shall be paid by the Licensee in the following manner:

       A.)  Fifteen days after the end of each calendar month commencing with the month after the Closing Date, Licensee shall pay Licensor the described percentage of such prior month's product sales attributable to Licensor's distributor organization.  Any such payment shall be past due if not received by Licensor before the 30111 day of such month.

       B.) If any inventory is transferred to Licensee on a consignment basis, Licensor shall ship its entire remaining inventory to Licensee on consignment , along with a list of such inventory shipped and the inventory cost.  Fifteen days after the end of each calendar month conunencing with the month after the Closing Date, Licensee shall pay Licensor the cost, as listed on the inventory provided to Licensee, of all inventory sold the prior month which was on consignment.  Each month, Licensee shall also provide a monthly report of inventory sold and inventory remaining on hand.

       C.)  Commission payments shall follow Licensee's regular business cycle, which currently is payment of commissions once per month on the fifteenth (l5th) of each month.

    4.  Term.  The nature of this Agreement is to last for the life of Licensor.  However, the following issues may result in termination of this Agreement if not cured within thirty (30) days and such cure must meet the approval of the offended party.

       A.)  If Licensor materially breaches paragraphs 6, 7, 8, and/or 9, and is not cured to the approval of Licensee, Licensee shall be able to terminate this Agreement and completely retain the rights to Licensor's products, inventory, distributor organization and other assets described within this Agreement.

       B.)  If Licensee materially breaches paragraph s 6, 10, and/or 11, and is not cured to the approval of Licensor , Licensor shall be able to terminate this Agreement and recover such damages as described in this Agreement.

    5.  Closing.  Licensor agrees to close this transaction on March 31, 2012. (the "Closing Date").

    6.  Post -Closing Obligations of Licensor/Licensee.

       A.)  Licensor agrees to use its best efforts in assisting with the transition I transfer of the Licen sor's distributor/customer organization and records to Licensee.  After Closing, J,icensor agrees to refer all order and inquiries related to the subject matter of this Agreement to Licensee.  Licensor further agrees to allow reasonable access to historical financial information as necessary.

       B.)  Licensee agrees to allow Licensor reasonable access to its books and records for audit purposes of the net product sales attributable to the Licensor's distributor organization.  Licensee agrees further to reasonably cooperate with Licensor's requests for information. Licensor shall notify Licensee no Jess than ten (10) business days prior to the date of its intended inspection of books and records.  Such information shall be confidential and Licensor shall not disseminate such info1mation and may only use it for audit purposes.

       C.)  Licensee agrees to use its best efforts to maintain and increase the sales of the Licensor's distributor organization exclusively  licensed from Licensor .  Licensee further agrees not to materially alter, transfer , assign, or otherwise dispose of such distributor organization until payment in full and the maturity of this agreement.

 

  

-2-

  

 

       D.) Licensee shall use its best efforts to sell the inventory acquired from Licensor and to prioritize the sale of such inventory.

       E.) Licensee and Licensor acknowledge that Licensor's distributor organization , inventory, and the other assets constitute valuable assets of Licensor 's. Licensee and Licensor therefore agree that during the tem1 of this Agreement and for two (2) year period after this Agreement, Licensors shall not contract with, utilize or attempt to utilize whether directly or indirectly, any portion of the either Licensor's and/or Licensee's distributor organizations, if such action could have the effect of re-directing the resources and skills of such distributors or distributor organizations . Such action would have serious and undeterminable financial ramifications that Licensor could be held responsible for.

 

    7.  Covenants of Licensor. Licensor covenants to Licensee that Licensor has good and marketable title in and to the assets being licensed and/or sold, free of all debts, liens and encumbrances, except as is expressly provided for herein.

    8.  Licensor's Representations and Warranties. Licensor represents and warrants to Licensee the following matters as of the date hereof, each of which shall also be true and complete as of the Closing Date as if made on the date of Closing, and each shall be deemed to be independently material:

       A.) That Licensor is a Limited Liability Company (LLC) duly organized , validly existing and in good standing under the laws of the state of California. That all signatures to this Agreement have full and official power to carry on the business as it is now being conducted and to enter into this Agreement on hehalf of Licensor and to bind Licensor to the terms thereof.

       B.)  That the execution of this Agreement by Licensor and its delivery to Licensee have been duly authorized by Licensor's Board of Directors and such Agreement and the execution and delivery thereof have been duly approved by all the holders of Licensor's outstanding shares; and no further corporate action is necessary on Licensor's part to make

this Agreement valid and binding upon it in accordance with its terms.

       C.)  That the assets being sold to Licensee arc free from all security interests, mortgages , liens, claims, defects of title, and encumbrances, save and except set out on the Schedule of Contracts and Liabilities appended  hereto .

       D.) That Licensor is neither a foreign corporation , foreign person nor intermediary for a foreign corporation or foreign person, subject to withholding requirements of the Internal Revenue Service.

    9.  Breach of Representations and Warranties. In the event that any of the above representations or warranties are breached , then Licensee shall have the right to recover its damages from Licensor.

    10.  Licensee's Representations and Warranties.   Licensee represents and warrants as follow, to-wit:

       A.) That Licensee is a corporation duly organized, validly existing and in good standing under the laws of the state of Delaware and has full corporate power to carry on its business as it is now being conducted and to enter into and fully perform its obligations under the term of this Agreement.

       B.) That the proprietary information relating to the distributor organization made the subject of this Agreement shall remain confidential and neither Licensee nor Licensor shall release or otherwise disclose, except as required by law, such information without the prior \Witten consent of the other party.

       C.) That Licensor will have the right to purchase True2Life product inventory of up to 500.00 per month at the product cost (cost of each item to Youngevity) for the purpose of personal use, donation, and business building activities such as sampling of the products to others. There will be no royalties or license fees paid on these items when purchased.

       D.) Licensee shall advance up to $4,000 dollars to be used to pay Licensor's software vendor for suppo1i of the Licensor's software systems.

 

  

-3-

  

 

    11.  Default. If Licensee fails to timely pay or fails to perform any of Licensees obligations or breaches any of Licensees representations or warranties , Licensor may recover its damages, elect to rescind this Agreement and/or seek any and all equitable or legal remedies available.

    12.  Other Documents.  Licensor further agrees to execute and deliver to Licensee all deeds, assignments, documents of title, and other instruments which may be necessary to effect the transfer of the assets and properties described in this Agreement.

   

    13.  Notices.  Any notice, request, demand, instruction or other communication to be given to either party herew1der, except for those required to be delivered at Closing, shall be in writing and shall be deemed to be delivered upon the earlier of actual-receipt (whether by hand delivery or delivery service) or upon deposit with the U.S. Postal Service, registered or certified mail , postage prepaid, return receipt requested , and addressed as follows:

                If to Licensor:

 

                _____________________

                _____________________

                _____________________

                If to Licensee:

                Youngevity

                Attn: Steve Wallach

                2400 Boswell Rd.

                Chula Vista, Ca 91914

    14.  Governing Law / Jurisdiction. This Agreement is being executed and delivered, and is intended to be performed, in the State of California, and the laws of such State shall govern the validity, construction, enforcement and interpretation of this Agreement unless otherwise specified herein.

    15.  Entirety and Amendments.  This Agreement and the Exhibits attached hereto embody the entire agreement between the parties and supersedes all prior agreements and understandings, if any, relating to the Subject Property and may be amended or supplemented only by an instrument in writing executed by the party against whom enforcement is sought. Further, the prevailing party in any litigation between the parties shall be entitled to recover, as a part of its judgment  reasonable  attorney's fees.

    16.  Invalid Provisions.  If any provision of this Agreement is held to be illegal, invalid or unenforceable  under present or future laws, such provisions shall he fully severable and this Agreement shall be construed and enforced as if such illegal, invalid, or unenforceable provision had never comprised a part of the Agreement. The remaining provisions of the Agreement shall remain in full force and effect and shall not be affected by the illegal, invalid or unenforceable provision or by its severance from this Agreement.

Furthermore , in lieu of such illegal, invalid or unenforceable provision , there

    17.  Parties Bound. This Agreement shall be binding upon and inure to the benefit of Licensee and Licensor and their respective heirs, personal representatives, successors and assigns.

    18.  Further Acts.  In addition to the acts recited in this Agreement to be performed by Licensor and Licensee, Licensee and Licensor agree to perform or cause to be performed at or after Closing any and all such further acts as may be reasonably necessary to consummate the sale contemplated hereby.

    19.  Third Party Beneficiaries.  The rights, privileges, benefits and obligations arising under or created by this Agreement are intended to apply to and shall only apply to Licensee and Licensor and no other persons or entities.

 

  

-4-

  

 

    20.  Licensee's Authority. The person executing this Agreement on behalf of Licensee warrants to Licensor that he has the Authority to execute this Agreement on behalf of Licensee and to bind Licensee pursuant to the terms hereof.

 

    21.  Effective Date.  The effective date of this Agreement shall be the date this Agreement is executed by both Licensor and Licensee. References to "Date of Agreement " are to the effective date.

    22.  Captions.  The captions herein contained are for the purpose of identification only and shall not be considered in construing this Agreement.

    23.  Time is of Essence. Time is of the essence of this Agreement and each and every provision hereof.

    24.  Assignment.  Licensee may assign its right, title and interest in and to the agreement to any person or entity, upon approval of Licensor.

    25.  Arbitration / Attorney's fees.  Any controversy or claim arising out of or related to this Agreement or its subject matter, or the breach thereof, shall be settled by arbitration in accordance with the Commercial Arbitration Rules of the American Arbitration Association ("AAA"), but not administered by the AAA , before a panel of three Arbitrators whose compensation therefore shall be set by agreement of the parties should such proceeding become necessary. The panel shall be selected by each party selecting one neutral arbitrator and the persons thus selected shall select a third arbitrator who may not be a person suggested by either pa11y . Judgment upon the award rendered by the arbitrator may be entered in any Court having jurisdiction thereof. Any Arbitration shall be conducted in San Diego, California. The non-prevailing party in any cause of action brought hereunder, pursuant hereto, or in co1mection herewith, inclusive without limitation of an action for declaratory or equitable relief, shall be liable for the reasonable attorney's fees, expenses and costs of suit incurred by the prevailing party therein.

 

EXECUTED by Licensor this __________ day of __________, 2012

 

Licensor

 

_____________________

By:  __________________

 

EXECUTED by Licensee this 31 day of March, 2012

 

Licensee:

 

AL International, Inc.

 

By:  /s/ Steve Wallach

Steve Wallach, Chairman and CEO

 

  

-5-

  

 

or equitable relief, shall be liable for the reasonable attorney's fees, expenses and costs of suit incurred by the prevailing party therein.

 

EXECUTED by Licensor this __________ day of __________, 2012

 

Licensor

 

 /s/ Luis Arriaza 

 By:  Dr. Luis Arriaza 

 

EXECUTED by Licensee this 31 day of March, 2012

 

Licensee:

 

AL International, Inc.

 

By:  /s/ Steve Wallach

Steve Wallach, Chairman and CEOEXHIBIT 10.6

   
 Exhibit 10.6
 Research Plan
 University of Arkansas for Medical Sciences (UAMS) and Cyto Wave Technologies Inc.
 

 TITLE: Optimization of an experimental prototype for photoacoustic detection of circulating melanoma cells and clinical trials in humans.
 

 1. INTRODUCTION
 Despite our substantial efforts to understand the biology of cancermetastases, which causes up to 90% of cancer-related deaths, especially melanoma, are still poorly understood.  Moreover,  no early metastasis diagnostic tests are currently available. Comprehensive studies have demonstrated the potential of using circulating tumor cell (CTC) count as a marker of metastatic development, cancer recurrence, and therapeutic efficacy.  A variety of assays have been developed to detect CTCs in peripheral blood samples ex vivo (see below).  However, due to a sampling problem, the sensitivity of these assays are limited (≥ 1-5 CTCs/mL), and incurable metastasis is already established at the time of the initial diagnosis.  To ensure the presence and isolation of rare CTCs, relatively large blood volumes are required.  
 This problem can be overcome by using in vivo photoacoustic (PA) cytometry (PAFC) technology pioneered by Dr. Zharov in 2006. The principle of PAFC is based on laser irradiation of the selected blood vessels using short laser pulses followed by time–resolved detection of laser-induced acoustic waves from CTCs (referred to as PA signals), with an ultrasound transducer gently attached to the skin area above the blood vessels. The mechanism of PAFC is based on the PA effect associated with fast non-radiative relaxation of absorbed laser energy in melanin particles as an intrinsic absorber in melanoma cells, followed by the thermal expansion of heated melanin particles which leads to thermoelastic generation of acoustic waves. 
 

 2. GOAL 
 The aim of this research is to develop a clinically-relevant, portable, experimental prototype for real-time, label-free photoacoustic (PA) detection of circulating tumor cells (CTCs) directly in the bloodstream of melanoma patients. Melanin will be used as intrinsic melanoma markers.  We will accomplish the first phase of this project by designing and optimizing the main parameters of this prototype with a focus on optimal laser characteristics, fiber-based delivery of laser radiation to selected vessels, the focused ultrasound transducers, the location of the portable PA probe on the skin, the position of the patient, the necessary software, and independent verification of PA data in vitro with conventional and new melanoma CTC assays.  This optimization will provide the maximal signal-to-noise ratio, assuming maximal PA signal from CTC, and minimal background noise in the PA signal from red blood cells (RBCs) in a minimized detection volume.  After testing the prototype on the 10 healthy volunteers, this prototype will be used for real-time monitoring in vivo in human blood from approximately 60 melanoma patients. 
   
 3. CURRENT ASSAYS FOR DETECTION OF CTC AND THEIR LIMITATION 
 Currently, various advanced assays are used to detect CTCs in a sample (1–10 mL) of peripheral blood, including: reverse transcription–polymerase chain reaction (RT-PCR), optical detectors, microfluidic chip techniques, and Cell SearchTM (Veridex LLC).  Combined with specific cell-enrichment techniques, these methods have sensitivities of 1–5 CTCs/mL. Some difficulties in reproducing the results of the preferentially used RT-PCR assay (e.g., for melanoma) are associated with differences in sample processing and the generation of false-positive signals due to contamination events, amplification of pseudogenes, and illegitimate transcription.  False-negative signals, in contrast, are related to the poor quality of source materials, intermittent shedding of CTCs into the bloodstream, and the genomic instability of malignant cells.  Further, RT-PCR is an indirect method and cannot provide direct evidence of the presence of intact CTCs in blood.  
 A principal drawback of all the other currently available CTC cyto-assays is that they are performed in vitro only, and hence their sensitivity threshold is limited by the sample volume. The ultimate threshold cannot be better than one CTC per sample volume (i.e., 31 CTC/mL for a 1-mL sample), which is equivalent to 35,000 CTCs in an adult patient’s blood volume of ~5 L. Recent data indicate that this CTC amount is sufficient for the development of metastases, especially in aggressive melanoma that metastasize at a very early stage. It might be considered too late to treat patients, and hence it is difficult to improve their survival when incurable metastases are already present at the time of the initial diagnosis using the current assays. Low sensitivity of 
 

 1
 

 

 
 current assays may also prevent monitoring the efficacy of therapy when the number of CTCs is reduced to £1 CTC/mL. The existing cancer blood test is time-consuming, taking at least a 5-6 hours to 1 day to perform, and can only screen cells at the site of venipuncture (e.g., normally the cubital vein) at limited, discrete time points.  This does not permit continuous monitoring of CTCs in a manageable timeframe to improve chances of patient survival. There currently are no well-established assays for assessment of melanoma CTCs both in vitro and ex vivo. 
 In addition, methods of cell manipulation, separation, isolation and enrichment followed by molecular cancer cell profiling, revolutionized cancer diagnosis and therapy. Various physical cell properties, including size, motility, electrical dipole moments, as well as optical and magnetic qualities have been exploited for this purpose. In particular, cells in biological fluids such as blood, urine, or cerebrospinal liquids, were labeled using magnetic microbeads and nanoparticles (NPs), and then they were separated and enriched from the sample flow by a magnetic field. To date, these techniques are used only in ex vivo, and thus the number of isolated CTCs was limited by the sample volume (see above). 
 Most of these problems can be solved by assessing blood in vivo, using methods Dr. Zharov has been pioneering and developing since 2004. It should be noted that there are several PA systems on the market, and approximately 10 additional research setups which are currently using PA effects. However, most of these systems are designed for PA imaging, microscopy and 3D tomography, and all of them cannot be used for detection of CTCs because there are limitations either in speed, or in sensitivity. 
   
 4. PRIORY STUDY OF PAFC CAPILITY TO DETECT CTC. 
 The principle of in vivo PAFC is based on the irradiation of selected vessels using short (nanosecond) laser pulses followed by time–resolved detection of laser-induced acoustic waves (referred to as PA signals) with an ultrasound transducer pressed against the skin (Fig.1). Most pre-clinical studies were performed on well-distinguished, 30-50-μm diameter blood vessels located approximately 40-100 μm deep in the thin ear vessels (250-280 μm) of the nude mouse model. Laser radiation can be delivered to biotissue either by using a microscope schematic with a customized condenser to create the desired linear beam shapes (e.g., from 5 ́50 μm to25 ́150 μm), or a fiber with a miniature tip and cylindrical optics. 
 	
	 

	 Fig. 1 Schematic of in vivo PAFC.  

 

 PAFC molecular specificity is provided either by label-free, intrinsic, absorption spectroscopic contrast (e.g., from hemoglobin [Hb], and melanin), or by strongly absorbing, low-toxicity, functionalized nanoparticles (NPs). 
 

 We demonstrated that PAFC has the potential for label-free detection of melanoma CTCs with the possibility of simultaneously eradicating them by a photothermal (PT) ablation mechanism, using PA CTC counting to control the efficiency of the PT therapy.  Specifically, we selected cutaneous melanoma as an almost ideal cancer for PAFC technology to provide routine, label-free, in vivo clinical assessment of CTCs for earlier detection of the most aggressive and epidemically growing malignancies which often progress to incurable metastasis at a very early stage of the disease. The label-free nature of PAFC when applied to melanoma denotes that PAFC can be translated to clinical application at a much earlier stage for melanoma than for other cancers, with obvious and beneficial public-health consequences for this devastating disease. On tumor (B16F10)-bearing models (Fig.2A-B) we revealed that CTCs  appeared in ear microvessels near the tumor on Week two, with no cells detected in the abdominal skin blood vessels. Three weeks later, CTCs appeared in the systemic circulation. This indicates a much greater likelihood of detecting the initial metastatic process in the vicinity of the primary tumor before CTCs are disseminated into the large blood pool. The skin tumor growth rate was faster than that of ear tumors, and CTCs also appeared more quickly in the circulation. In particular, by Week one, 1–4 CTCs/min were detected in the skin vasculature, and as the tumor size increased, the number of CTCs gradually increased (Fig. 2C) to ~7 CTCs/min and ~12 CTCs/min by Week 3 and Week 4, respectively. On the occasion, either PA signals with complex shapes, or one large PA signal were observed, which support the hypothesis of circulating melanoma cells as aggregates. Indeed, optical imaging of ear vessels near the tumor revealed CTC aggregates on the vessel wall, indicating a high probability of CTC aggregating during intravasation. 
 

 2
 

 

 
 

 	
	 

	 Fig. 2 In vivo label-free, PA detection of melanoma CTCs. Melanoma tumor growth in the mouse ear (A) and skin (B). D) Change in the CTC count in the microvessels of the abdominal skin as a function of time after B16F10 tumor cell inoculation in the ear (red empty circle) and skin (blue empty square).  The dark red circle and blue square indicate averaged data. Laser parameters: wavelength: 904 nm; pulse energy fluence: 30 mJ/cm2; pulse rate: 10 kHz).

  
 The mice were euthanized, and tissue sections from different organs (e.g., lung, liver, brain, or lymph nodes) were examined by immunohistochemical staining. No evidence of metastasis was found during the first 3 weeks after tumor inoculation for the ear tumor mouse model, while PAFC demonstrated early detection of CTCs 4 days after tumor inoculation. Thus, CTCs can be readily detected with PAFC weeks before any evidence of detectable metastasis appears in tissue samples using conventional techniques. 
 By using PA to count rare CTCs, we estimated the PAFC’s sensitivity threshold as 0.5-1 CTCs/mL. This was verified ex-vivo by assessing the whole blood volume using scanning PT and PA cytometry. This unprecedented threshold sensitivity on the mouse model provided an opportunity to use PAFC as a powerful research tool to study CTC behaviors and CTC’s role in metastasis development at an early stage of cancer development. The PAFC sensitivity has the potential to be further improved 102-103 -fold (i.e.,~1 CTCs/100mL or 1 CTCs/1000mL)  by the examination of a larger blood volume in humans, which is unachievable using any existing assays.
 We believe that the PAFC technology may have a tremendous clinical significance due to its high sensitivity. It can indicate the presence of CTCs in the blood at an extremely low concentration, much below the sensitivity threshold of other methods.  Clinical applications may include: 1) blood screening for early CTCs before metastases progression; 2) testing for cancer recurrence; 3) individualized assessment of the therapeutic intervention (e.g., surgery, chemo, or radiation) and its efficiency through real-time CTC counting; and 4) potential for metastasis inhibition, or prevention by using a well-timed therapy. In particular, when we exposed an abdominal vessel to an 820-nm- wavelength laser, increasing the energy fluence from 60 mJ/cm2 to 600 J/cm2 led to an increase of PA contrast of the CTCs above that of the RBC background by ~6 times. This phenomenon was associated with laser-induced nanobubbles around overheated, strongly absorbing melanin nanoclusters in melanoma cells, which served as a nonlinear PA signal amplifier compared to linear PA signals from RBCs with a homogenous hemoglobin distribution (i.e., with no nanobubble formation). On the other hand, the rate of CTCs gradually decreased from 12 CTC/min to 1-2 CTC/min over a one hour monitoring period. This effect was also associated with the generation of nanobubbles, not only as a PA signal  amplifier, but also at a specific laser energy as a melanoma cell killer. These data demonstrated an important potential to use PAFC to guide blood purging in vivo by periodically exposing the blood vessels to the laser. Further study could determine whether this new treatment is effective enough to be used alone, or whether it should be used in combination with chemo-or radiation therapy
 For many years, oncologists believed that some medical intervention may provoke metastasis; however, no direct evidences were previously presented.  Using the techniques and animal models described above, we discovered that palpation, biopsy, conventional and laser surgery may either initiate CTC release in the blood which previously did not contain CTCs, or may dramatically increase (10-50–fold) the CTC counts above the previous level, which can increase the risk of metastasis.  In particular, the ~100 g weight pressure or palpation  (by squeezing of melanoma tumor with fingers), notably increased the CTC count that eventually led to the appearance of lung metastasis at Week 3.
 

 5.  SUMMARY/CONCLUSION 
 Our previous findings have demonstrated, for the first time: (1) PAFC at its current stage of development provides label-free noninvasive detection of melanoma CTCs; (2) that there are potentials to further improve PAFC’s  parameters by a non-linear signal amplification; (3) PAFC’s potential to achieve sensitivity of 1 CTC/10 mL in humans (i.e., approximately one order better than in existing assays) during ~1hr by monitoring of 200-300 mm vessel with focused linear laser beam, sensitivity of 1 CTC/100 mL; by examination of 1-3 mm 
 

 3
 

 

 
 peripheral blood vessels  at depths of 1-2 mm and 1 CTC/1 L; and by examination of 1 cm vessels (e.g., jugular vein or carotid artery) at depths of 0.6-1,5 cm 1-2 mm with a monitoring time of only a few minutes. 
 Our preliminary studies, however, had the following limitations that the proposed work will overcome: (1) we demonstrated only the feasibility of the concept of PAFC on the animal model; and (2) the clinical prototype with the optimized PAFC parameters has not yet been developed.  To maximize the sensitivity of PAFC, the tasks of achieving the best optical and acoustic resolutions have to be resolved. 
 

 6. RESEARCH PLAN UNDER AGREEMENT (UAMS AND CYTO WAVE INC.) 
 

 Goal. The objective of this proposal is to develop a clinically relevant portable PAFC prototype with fiber-based PA probes for in vivo label-free detection of pigmented melanoma CTCs. The expected sensitivity in the label-free mode is as low as 1 CTC/ml of blood in the animal model and ~1 CTC/1 L in humans.  This goal will be accomplished through the Specific Aims below. 
 

 The results of the project will be used to enhance the sensitivity of the clinical prototype, increase its safety by decreasing laser power requirements, simplify system alignment and, probably, allow real-time refocusing of the probes to account for light scattering in tissues. In addition, successful completion of these steps would provide optimal system performance over time and would simplify the use of the system by nurse personnel.  
 

 PROJECT TIMELINE 
 Aim 1:  Optimize of PAFC parameters, months 1–5. 
 Aim 2:  Optimize and incorporate software in PAFC, months 1–5. 
 Aim 3:  Optimization of PAFC for clinical conditions, months 4-5.
 Aim 4:  Develop and/or test CTC assays in vitro (for verification PA data in vivo), months 1-5. 
 Aim 5:  Test PAFC prototype on 10 healthy volunteers and 6-10 melanoma patients, months 3-5 (according to the IRB protocol). 
 Aim 6:  Test PAFC prototype on approximately 60 melanoma patients, months 7-12 (according to the IRB protocol) 
 

 4

Source: [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00216-of-00352.parquet"}, [{"source": "alea-institute/alea-institute/kl3m-data-edgar-agreements/train-00216-of-00352.parquet"}]]