Biomedical Discoveries via ROV
A mustard yellow sponge seen off western Puerto Rico at a depth of approximately 75 m (246 ft.) during the Exploration of Deepwater Habitats off Puerto Rico and the U.S. Virgin Islands for Biotechnology Potential expedition.
By Dr. Esther Guzmán • Samantha Flounders
Sixty-two percent of current medicines have their origin in natural products. Natural products are small molecules made by a living organism. Since the oceans cover more than 70 percent of the Earth’s surface and contain a wealth of biological diversity, it is logical to expect that many discoveries can come from the ocean. Many marine organisms have genes that are similar to those in humans, although they are used for different functions. Similarly, certain processes are shared between humans and marine organisms.
Drugs from the sea are slowly turning into a reality: Ara-C or cytarabine has been approved since 1969 to treat leukemia; Halaven is the synthetic version of eribulin mesylate, a natural product obtained from a marine sponge that was approved in 2010 to treat metastatic breast cancer; Yondelis is the semi-synthetic version of the natural product from a tunicate and was approved in 2015 to treat soft tissue sarcoma and ovarian cancer.
More recently, antibody-drug conjugates (ADC) are seeing clinical success. ADCs combine a natural product that kills cancer cells with an antibody to guide it to the tumor. Polivy, approved in 2019 to treat lymphomas; PADCEV, approved in 2019 to treat metastatic urothelial cancers; and TIVDAK, approved in 2021 to treat metastatic cervical cancer, use monomethyl auristatin E (MMAE), a synthetic version of the natural product made by a cyanobacteria, to kill the cancer cells.
HBOI and UNCW UVP Partnership
The Marine Biomedical and Biotechnology Research Program at Harbor Branch Oceanographic Institute (HBOI; a part of Florida Atlantic University since 2007) has been doing drug discovery from marine organisms since 1984, as an added value to the ocean exploration conducted with the Johnson-Sea-Link (JSL) manned submersible. This submersible had outstanding collecting capabilities, the ability to hover, and reached depths to 1,000 m. Ocean exploration with the JSL led to a collection of about 30,000 invertebrates and about 19,000 micro-organisms related to drug discovery work.
The selling of the institute’s ships in 2010 and the retirement of the submersible in 2011 hampered how the group combined ocean exploration with drug discovery. The need to recollect organisms that contain compounds of interest and the desire to continue the exploration of the oceans for medicinal purposes led to looking at alternative forms of collecting. The solution was found in the Mohawk ROV, a sophisticated, versatile vehicle with high reliability and mobility. The ROV has a working depth of 1,000 m and is maintained and operated by the University of North Carolina Wilmington (UNCW) Undersea Vehicles Program (UVP), which comprises three ROV pilots and technicians. The small, dedicated team operates the Mohawk ROV from vessels of opportunity, and this can support a wide variety of research missions.
A white sponge seen off northwestern Puerto Rico at a depth of approximately 111 m (364 ft.). The green and red lasers are from two different cameras, and the distance between them measures 10 cm. This allows scientists to determine the size of a sample for collection. The large tubular sponge is an Aplysina, and this class of sponge is known to have compounds with anti-cancer activity, including activity against triple-negative breast cancers.
Mohawk ROV
The Mohawk ROV is outfitted with a custom-designed, science-specific tool skid developed by HBOI and modeled after the JSL manned submersible. The skid powers scientific instruments, such as the Sea-Bird SBE 49 Fast-CAT, a CTD sensor, and the PyroScience AquapHOx-LX O2 and pH sensor, an oxygen long-term logger. Additional scientific sensors may be added, depending on the scientists’ data collection objectives.
The Mohawk’s science skid is also equipped with an ECA Hytec Micro 5E, a five-function all-electric manipulator arm. Samples can be picked up, snipped or suctioned. The manipulator and suction sampler provide the ability to collect a wide variety of sample sizes, textures, and consistencies. All samples are georeferenced so that the ROV can return to the exact spot in the future if necessary.
The Mohawk ROV captures high-definition (HD) video throughout the entirety of the dive with an Insite Pacific Mini Zeus Mark II color video camera with 10:1 zoom, auto/manual iris, auto/manual focus and 2.38 million effective pixels. This high-definition video with extreme zoom capabilities provides the science party with a clear, detailed, real-time view of the underwater environment.
The Mohawk’s compact frame and agile mobility allows for the pilots to fly the vehicle right up to prospective samples for a closer look. Digital still photography is controlled by the science party and captured by a Kongsberg Maritime OE14-408 10-mp digital still camera with 5x zoom. Digital still photos are paired with an OE11-442 strobe with evaluative through-the-lens (E-TTL) auto flash metering, which calculates how much light the flash needs to emit for appropriate brightness. The video camera, stills camera and strobe are mounted to a powered tilt platform, which allows for proper framing of an image.
It is of the utmost importance to capture and record the samples in situ. Once the science party decides they would like to acquire a sample, a digital still photograph is properly framed and zoomed as desired. Every photo and all video are laser-scaled at 10 cm by dual lasers mounted to the digital still camera and video camera. This provides a quick size reference via two laser dots when looking at sample photos. Underwater imaging provides the visual record of how the samples truly existed in their natural environment. Once they are brought to the surface, they can look very different. Some organisms release substances (such as mucus and inks) that can change their appearance when sub-sampled.
Furthermore, underwater imagery provides a visual timeline for monitoring reef health and documenting changes over time. Habitat characterization over time is of critical importance for understanding the health of our oceans.
Many times, no sample is necessary, and the agile maneuvering of the Mohawk ROV allows for the pilots to fly and land in densely populated areas for a high-definition look and take framed, scaled, and zoomed photos.
Flower Banks Expedition
An expedition to the Flower Garden Banks National Marine Sanctuary (FGBNMS) in 2022 funded by NOAA Ocean Exploration (OER) led to the discovery and documentation of rich biodiversity outside of the sanctuary’s protected areas. The Mohawk ROV was the ideal vehicle for the mission, collecting more than 175 samples in just nine days. The pilots and manipulator operators of the Mohawk are known for their capability to fill up the collection skid repeatedly on multiple bounce dives in a day. While only a few samples have been tested for their therapeutic potential, there are already some leads.
The process of extraction is similar to making coffee. Whenever you make coffee, you extract the chemicals in the coffee beans using hot water, which include about a thousand chemicals that give coffee its taste and smell. Instead of extraction via hot water, marine organisms are extracted using alcohol, then separated into mixtures of four to five compounds that are used to screen for activity.
Some forms of pancreatic, lung, breast, skin and colon cancers have mutations that activate unusual pathways to obtain nutrients that allow these cancers to grow. About 575 samples from organisms collected during the FGBNMS cruise were screened for their potential to inhibit these unusual pathways from obtaining nutrients, with 99 samples corresponding to 37 organisms shown to have this activity. These compounds could help in the treatment of the cancers mentioned.
Triple-negative breast cancer receives its name because it lacks the estrogen, progesterone and epidermal growth factor 2 receptors on its surface. This lack of receptors makes these cancers difficult to treat. Although they only represent about 12 percent of all breast cancers, they tend to be aggressive. About 234 samples corresponding to 10 organisms collected during the FGBNMS expedition have been tested for their potential to selectively kill triple-negative breast cancer cells when grown as spheroids, or little tumors, with 52 samples exhibiting this activity. We are very excited about these samples because their mode of action may be different from known drugs, adding potential new ways to treat this disease.
Extracts from bacterial samples obtained from the samples collected during this expedition were also tested for their ability to be novel antibiotics. About 313 samples were tested for their potential to inhibit the growth of multidrug-resistant Staphylococcus aureus (MRSA), an infection that is prevalent in many hospitals; 79 samples exhibited this activity. Two-hundred twenty-four samples were tested for their potential to inhibit the growth of the yeast Candida albicans, with 21 exhibiting this activity.
The Mohawk ROV sits on deck after a dive. The Mohawk ROV was used to collect sponge and coral samples, as well as video and still photos, in two recent expeditions.
Ongoing and Future Work
The activities highlighted above were the ones found in-house using our current assays from a limited number of organisms. More samples will be added to the library to make them available for broader screening, increasing the chance for more leads.
In the past, samples from our collection have been tested against other diseases, such as Alzheimer’s, and cancers through collaborations with other universities. Moreover, as new assays become available in-house, samples from the library are tested in these, resulting in finding new activities from compounds that were already known. (HBOI collections are also studied to support taxonomic, ecological, evolutionary and climatological research conducted by scientists worldwide through collaborations.)
Earlier this year, in a NOAA OER-funded expedition, HBOI used the Mohawk ROV to explore the deepwater habitats off Puerto Rico. The Mohawk effectively and efficiently collected 136 samples for biotechnology studies. All of us are eager to see what activity against dreaded diseases these samples may possess. HBOI is enthusiastic to use the Mohawk’s new 1,000-m system in the future to expand the range of our biomedical treasure hunt.
Samantha Flounders is an ROV pilot and technician with the UNCW Undersea Vehicles Program.