Fake, fun and fantastic, myths usually tell us something about why the world is the way it is. But believing myths about cancer can be outright dangerous. Consider this: cancer is contagious and you need dollops of positivity to beat the dreaded C-word. Or this: cancer is caused by a fungus and its actually your bodys attempt to protect itself from the infection. From coffee to carbonates to cannabis, millions of pages on the internet tell us every day about miracle cures for cancer. And it is hard to distinguish fact from fiction, because few illnesses cause as much terror or have as many misconceptions that refuse to die. Here, some of the countrys leading oncologists bust old myths and bring new hope, as an explosion of new research changes the way cancer is perceived, understood and treated.

Dealing with cancer means making some tough choices. How you decide to seek medical help will determine your chances of cure. Hence, education is vital. The most common concerns patients come to me with are often some of the biggest myths. I find it painful that the myths are very much alive even among the educated classes. I make time to engage with my patients to dispel the misconceptions and to create awareness about the bad effects of these myths. Remember, you have a far better chance of beating the disease if you can dispel the fallacies and false notions.

Myth Biopsy can disturb cancer cells and cause them to spread to otherparts of the body.

Reality

It disturbs me that people still come to me and say, I dont want to do a biopsy. The truth is: there is little reason to worry that a biopsy allows cancer cells to spread within the body. Medical evidence shows that this is unlikely. In fact, patients who have biopsy procedures to confirm their diagnosis and determine the cancers stage have a better outcome and longer survival than patients who do not have a biopsy.

The point is, how can you diagnose a cancer without biopsy? Cancer treatment starts only after biopsy. It is the first landmark event in the definitive diagnosis of a cancer. That is crucial in determining and planning the correct treatment for a patient. If a biopsy indicates a benign disease, the patient is spared unnecessary treatments.

Biopsy may involve discomfort and bleeding, but the gain far outweighs the risks. I ask my patients if biopsy is responsible for cancer spreading, then all the people coming to me should have initial stage cancers, since they havent done biopsy as yet. I bring in their own examples: how come your own cancer has spread, as all the imaging, MRI, CT and PET scans show, although you have not done biopsy? Yet faith in this myth continues. It is a key reason why treatment gets delayed in our country. We have to understand that cancer does not wait for any procedure to spread.

MythChemotherapy means the cancer is terminal. Hence,avoid chemo.

Reality

The second set of cancer myths I come across revolves round chemotherapy. My patients ask a lot of questions about it: they want to know if chemotherapy means the illness is terminal, if the side-effects of chemo will completely disrupt their life completely, and if chemo is really worth it. A lot of patients do not wish to go through it.

The truth is: chemotherapy certainly does not mean a cancer is terminal. It is essential for many cancer patients. The more advanced cancer you have the more you need chemotherapy. In fact, chemotherapy is often a preventive measure to prevent recurrence and to improve the odds of cure. It also ensures faster recovery. Chemotherapy has evolved considerably over the decades. The drastic side effects are not as prevalent today. It does not disrupt a patients everyday life the way it used to once. With the advances in chemotherapy, more and more people are surviving and doing well after cancer treatment.

We need to understand that cancer is something that happens in all multicellular organismsfrom the smallest hydra to the biggest whales. Cancer is more common in animals that are domesticated or in the zoo, because they live longer than those in the wild. Studies from India have analysed fossils of dinosaurs that suffered from cancer. Cancer is a disease of cell regulation. In multicellular organisms there are many cells, some of which become bones, some tissues, some the brain. In that process of evolution, things can go wrong. And sometimes they can become uncontrollable. Cancer is such unchecked cell growth.

More recent studies show cancers are caused by three broad groups of cell alterations: the smallest group is that of people born with some genetic alteration that runs in the family. It makes up about 5 percent of all cancers. Of the environmental factors, the biggest is tobacco, which causes damage to the DNAcigarettes and passive smoking, chewed tobacco, hookah, dant manjanmaking up 25 percent of cancers. The next big group is infection, which is coming down now as hygiene is improving. The big ones are papilloma virusa sexually transmitted disease, from the male to the femaleit takes about 30 years to manifest, from pre-cancer to cancer and then to metastatic cancer. In the liver you get Hepatitis B and C, from mother to child.

Hep C comes through transfusion, tattooing. Then there is the Helicobacter pylori, a waterborne bacterium that is usually acquired in early life. If one is exposed to other carcinogens, like salted meat, cigarette smoke and so on, it triggers the cancer. Infections are responsible for 15-25 percent of cancers in India.The rest of the cancers are now thought to be random occurrences. Our body is a continuous workshop of cell regulation, replacement, regeneration: when cells get damaged, old cells are removed and new cells grow. The skin, the bone marrow, the linings of the gut, bladders and the kidneyare changing continuously. Stem cells provide new cells to the body as it grows and replace those that are damaged or lost. They can divide over and over again to produce new cells. As they divide, they can change into the other types of cell that make up the body. And just sometimes, things go wrongat randomthat have carcinogenic potential. That means, anyone can get cancer. Call it plain bad luck.

MythCancer is fast turninginto an epidemicin India.

Reality

That to me is the biggest myth. You may see a large number of cancer patients in hospitals. You may know a lot of people with cancer. But the epidemic is more apparent than real. Cancer is a disease of older age. It usually picks up from about age 40-50 and reaches a peak at about 7080. What has happened in India is that our population has exploded and we have a substantial number of older people. There are a 100 million people above age 60.

The number of cancer patients is also increased. This is typical of what we call an epidemiological transition. That means, once people died of infectious diseasestyphoid, malaria, dengue, choleraand so life expectancy was very low. In 1947, life expectancy was about 30-35 years. Now it is almost 70 years. So you dont just have more people, you have more older people.

Most importantly, we have started doing lab testsendoscopies, scans, screens, biopsiesso deep-seated cancers are being picked up. Even 30 years ago, there was no opportunity for this. This is why you are suddenly seeing so many people with cancer. The proof for this lies in some very beautiful studies done 100 years ago by British doctors in India. They had done autopsies on a lot of unclaimed bodies. And the autopsies showed a high rate of cancersalmost as high as it is today. The Mumbai Cancer Registry, which was started in 1962, has been monitoring cancers in the city continuously for nearly 60 years. You will be pleased to know that the rates have not really increased. The actual numbers have gone up, as the population of Mumbai has gone up enormously, but if you standardise the number of cancers in corresponding age groups and compare, there is no increase at all. On the contrary it has decreased a little bit, which is very reassuring.

MythAYUSH cancure cancer.

Reality

Every day in my chamber, I see patients who have been sitting on their symptoms for months, sometimes years, thinking it would not be cancer. Their doctors have not told them it could be cancer and treated them for dysentery or piles, instead. And when things get out of order, they come to oncologists. But by then, its often too late. There is a serious lack of awareness among people and among AYUSH practitioners. The bottomline is, if you have symptoms that continue for weeks, do not go to doctors who cannot do biopsy. You cant diagnose cancer without doing biopsy.

MythCancer is adeath sentence.

Reality

People panic once cancer is diagnosed. They seek out immediate treatment, go to people who are not experts, do not understand the upsides or downsides and end up getting treatments that harm them more. One reason could be that they are driven by the messages they hear. So the first thing to remember is that cancer is not a death sentence. Dont panic. You must do your research and find out people who are doing the right treatment and go to the right place. Cancer cells grow slowly, dividing every three to four weeks. It is not that scary. You have four to six weeks time to find the right place. The best treatments are inevitably found in places that have multidisciplinary teams.

Myth Cancers spread with intervention.

Reality

The biological nature of cancer is to spread. Thats why cancer kills. All cancers will spread depending on its tumour biology. Some fast and some slow. The reason why there is an apparent spread after an intervention via biopsy or surgery is because those procedures can reveal the spread more accurately, and not because cancers spread after the intervention. As the cancer cells divide in geometric proportions, it will take more or less time to double its size from 1 mm to 2 mm and from 5cm to 10 cm. In other words, the growth of cancer will appear to be faster in more advanced stages.

Myth Dont tell the patient the bad news.

Reality

If you tell the patient the bad news (diagnosis of cancer etc) they will not be able to take it. As a result, often half-truths or white lies are told by doctors to patients. Studies done in India on patients with cancer, however, reveal that 90 per cent of them want to know the correct diagnosis and likelihood of survival, as it helps to plan the treatment and plan their lives as well.

There are a whole range of rumours and myths about cancer that makes it hard for people to know what is true about this widely misunderstood disease. Every day, new myths arise and old ones reappear, leaving patients and family members confused and vulnerable. Many turn to dangerous remedies, others get trapped into believing fallacies that harm them immensely. Here are some of the most persistent and pernicious myths surrounding cancer and its treatment that we face every day.

MythNo, this cannotbe cancer.

Reality

We come across hundreds of patients who show signs and symptoms indicating the possibility of cancer, yet they will not accept it. The first reaction in India, typically, is: No, this cannot be cancer. Somebody gets a lump, a hard mass on any part of the body, a coughing spell that lasts for over four weeks. And the first thought is to blame it on infections or pollutionsomething that will settle down within a week or two. Who will tell them that cancer coughs will not settle down? The first presenting symptom for lung cancer will be cough. We get patients with telltale signs of cancerblood or discharge through urine, stool, mouth, female genital organsyet by the time they seek medical opinion, often the cancer has already spread to other parts of the body. This denial mode and not letting treatment start on time, is a very Indian attitude.

MythLifestyle tendencies that lead to heart disease, hypertension or stroke are notrelated to cancer.

Reality

Think of physical inactivity, of stress, of eating a lot of preserved and processed food. And the first reaction we find among patients is: Surely, those are not linked to cancer. They seem to be convinced that the lifestyle tendencies that are linked to heart disease, hypertension or stroke have nothing to do with cancer. Thats just not true. Now we have sufficient literature and scientific evidence to show that patients having more junk food, leading more sedentary lives, living in more stressful environments are more prone to cancer than those who are not under such circumstances. Fortunately, these are modifiable factors. That means, people can change these. Non-modifiable factors mean those that cannot be changed: for instance, somebody with a family history of cancer will have higher chances of getting the disease. These are genetic traits. There needs to be developed the maximum awareness that modifiable lifestyle excesses dont just bring on heart disease, stroke or hypertension, but also cancer.

MythCancer iscontagious.

Reality

This is an important myth that even educated people share. If somebody gets cancer, friends and neighbours do not visit, thinking they may also get it. This is a huge issue, because in India we are dependent on social support for emotional sustenance. That often breaks down for cancer patients, who are anyway emotionally disturbed and need more support. Myths like this make them go through social isolation, bringing in more stress. Cancer is absolutely not contagious. In some people, cancers may be caused by certain viruses (some types of human papillomavirus, or HPV, for example) and bacteria (such as Helicobacter pylori). While a virus or bacterium can spread from person to person, the cancers cannot.

MythNourishing foodnourishes cancer cells.

Reality

This is a common and dangerous misconception: if you give nourishing food to a patient, the cancer will grow faster. Hence deprive a cancer patient of food and the cancer will die out. People try to avoid taking nutritious food, become malnourished, their immunity gets weak and they are not able to tolerate chemotherapy, surgery, radiationfor all of which a patient needs to be physically as strong as possible. If they eat well, the immunity system of the body will fight the cancer cells. Remember, our body constantly makes some cells that can convert into tumour cells. At the same time, the body also has the immunity to destroy these cells. If immunity goes down, the patient becomes more prone to having cancer. Keeping yourself healthy and stress-free are the vital preventives for cancer.

THE COSTCancer is now the leading cause of catastrophic health spending, distress financing, and increasing expenditure before death in India

Out-of-pocket expenditure is three times higher for private inpatient cancer care in the country40% of cancer costs are met through borrowing, sale of assets and contributions from friends and relatives These costs exceed 20% of annual per capita household expenditure in 60% of Indian households with a patient with cancer

A lot of my patients ask: Why did I get cancer? I dont smoke, I dont drink, I am a vegetarian, I pray to God regularly, I have never harmed anybody. Then why did I get it? These are some of the most challenging questions oncologists face. And these are also at the intersection of the myths, misconceptions and facts in all discussions on cancer.

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This World Cancer Week, let's separate the myth and reality surrounding it - The New Indian Express

Abstract

Liver metastases often progress from primary cancers including uveal melanoma (UM), breast, and colon cancer. Molecular biomarker imaging is a new non-invasive approach for detecting early stage tumors. Here, we report the elevated expression of chemokine receptor 4 (CXCR4) in liver metastases in UM patients and metastatic UM mouse models, and development of a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, for sensitive MRI detection of UM liver metastases. ProCA32.CXCR4 exhibits high relaxivities (r1 = 30.9 mM1 s1, r2 = 43.2 mM1 s1, 1.5 T; r1 = 23.5 mM1 s1, r2 = 98.6 mM1 s1, 7.0 T), strong CXCR4 binding (Kd = 1.10 0.18 M), CXCR4 molecular imaging capability in metastatic and intrahepatic xenotransplantation UM mouse models. ProCA32.CXCR4 enables detecting UM liver metastases as small as 0.1 mm3. Further development of the CXCR4-targeted imaging agent should have strong translation potential for early detection, surveillance, and treatment stratification of liver metastases patients.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Approximately 50% of UM patients will develop metastases (1). About 93% of UM metastases occur in the liver, which results in death in almost all cases due to the lack of effective treatments (2). Through histological analysis of postmortem patient samples, UM liver metastases can be classified into three stages based on size (i.e., diameter): stage 1 (50 m in diameter), stage 2 (51 to 500 m in diameter), or stage 3 (>500 m in diameter) (3). Pathologically, UM hepatic metastases primarily have two growth patterns: infiltrative or nodular. The infiltrative pattern occurs when circulating metastatic UM cells lodge in the sinusoidal space and eventually replace the hepatic lobule. The nodular pattern metastases, however, originate in the periportal area. UM cells co-opt the portal vein, and when the tumor grows, it exhibits angiogenesis and effaces the adjacent hepatocytes (4).

There are major barriers facing clinicians in UM management, such as the lack of noninvasive and sensitive imaging methods for metastases, and the resistance of UM to traditional systemic chemotherapies (5, 6). Contrast-enhanced computed tomography (CT) is a widely used modality for screening of hepatic metastases (7); however, this method is not optimal for liver lesion characterization (8). 2-18F-fluoro-2-deoxy-d-glucose (18FDG) positron emission tomography/CT (PET/CT) not only can locate the hotspot for characterization of liver metastases but also has disadvantages due to the use of radiation dosimetry and the comparatively low specificity of the technology (9).

Magnetic resonance imaging (MRI) is the preferred clinical imaging modality for the assessment and characterization of liver malignancy because it does not use ionizing radiation and has high soft tissue penetration providing morphological, anatomical, and functional information. Dynamic-enhanced MRI, with liver-specific contrast agents, is widely used for liver lesion characterization, although its sensitivity and specificity are low for lesions less than 1 cm (10). In addition, MRI with the administration of clinically approved contrast agents can not differentiate the different growth patterns of UM metastases in the liver (11). Previous studies have demonstrated that molecular imaging of corresponding biomarker expression, such as HER2, improves detection sensitivity for cancers (12), but to date, diagnostic biomarkers for imaging UM liver metastases have not yet been established. Therefore, there is a pressing unmet medical need to develop MRI contrast agents for early detection and follow-up of liver metastases, especially for high-risk patients.

CXCR4 (chemokine receptor 4) plays a key role in cell migration and metastatic dissemination to several organs such as the liver, bone marrow, and lung, as these organs have intrinsically high concentrations of its natural ligand CXCL12 (Fig. 1A) (1315). A CXCR4 antagonist, plerixafor (Mozobil, AMD3100), has been approved by the U.S. Food and Drug Administration (FDA) for stem cell mobilization to the peripheral blood for autologous transplantation (16). CXCR4 expression has been proposed as a prognostic factor and a potential therapeutic target. Elevated expression of CXCR4 has been reported in several UM cell line studies (17, 18). Blockage of CXCR4 gene expression by transfection with CXCR4 small interfering RNA (siRNA) has been found to inhibit invasive properties of UM cells exposed to soluble factors produced by human livers (14). On the basis of these data, we hypothesized that CXCR4 would be a potential biomarker with treatment implications for imaging UM metastases in the liver.

(A) Tumor cells that express CXCR4 metastasize through CXCR4-CXCL12 interaction to specific organs that have intrinsically high concentrations of CXCL12 such as the lung, liver, and bone. (B) UM cell lines have elevated CXCR4 expression. Flow cytometry results measured elevated CXCR4 expression across different UM cell lines. Mel290 and M20-09-196 measured more than 80% of CXCR4 immunopositivity. Measurements of each cell line were done in triplicate. (C) CXCR4 IHC staining in liver tissue from metastatic UM patients (n = 4, IRS = 8.2 1.3). The liver metastases displayed strong red intensity, denoting strong CXCR4 expression. (D and E) CXCR4 IHC staining of primary UM (D) and hepatic metastases (E) in metastatic UM mice. UM hepatic metastases have higher CXCR4 expression compared with primary UM, indicated by the red staining. (F) CXCR4 IRS of primary UM and metastases in the liver in metastatic UM mice. Hepatic UM metastases displayed stronger CXCR4 expression (IRS = 9.5 0.8) than primary UM (IRS = 5.4 0.3). P 0.05.

In this study, we confirmed and validated that CXCR4 is a diagnostic imaging biomarker by its elevated expression in liver metastases in three different systems: ex vivo using samples of UM patients, in vitro UM cell lines, and in vivo mouse models. In addition, we have successfully designed a CXCR4-targeted, protein-based contrast agent, ProCA32.CXCR4, which can detect UM hepatic metastases as small as 0.1 mm3. The detected liver micrometastases were further validated by histological analysis, which correlated with MRI results. Our results indicated that ProCA32.CXCR4 enables precision MRI capable of defining molecular signatures for identifying metastases.

To validate CXCR4 as a biomarker for imaging UM metastases, we determined CXCR4 expression in multiple systems, including six UM cell lines, UM patientderived tissue, as well as a metastatic UM mouse model. Flow cytometry analyses of six UM cell lines revealed that CXCR4 is expressed across different UM cell lines. Among these, Mel290 and M20-09-196 cell lines exhibited more than 80% CXCR4 immunopositivity (Fig. 1B). Immunohistochemical (IHC) analysis of CXCR4 in UM patient liver tissue revealed that CXCR4 is highly expressed in liver metastases with both nodular and infiltrative growth patterns (Fig. 1C). We further observed elevated CXCR4 expression in primary ocular tumor and liver metastases in the metastatic UM mouse model generated by inoculation of M20-09-196 cells (Fig. 1, D and E), which have the BAP1 gene mutation that is often observed in aggressive UM liver metastases (19). In these M20-09-196 mice, the CXCR4 immunoreactive score (IRS) of UM metastases in the liver was significantly higher than in primary UM (P < 0.05, Fig. 1F). Together, these data indicated that CXCR4 expression is increased in UM metastases in the liver and may be a potential biomarker for diagnostic imaging of UM metastases.

Figure 2A presents the design of ProCA32.CXCR4 and the interaction of ProCA32.CXCR4 with CXCR4. ProCA32.CXCR4 was generated by engineering a CXCR4-targeting moiety into a protein contrast agent, ProCA32, which incorporates two designed gadolinium (Gd3+) binding sites (20). The viral chemokine analog viral macrophage inflammatory protein-II (vMIP-II) is encoded by the human herpes virus 8 and interacts with CXCR4. On the basis of the complex x-ray structure of CXCR4 and vMIP-II, we designed the CXCR4 targeting moiety, including key CXCR4 interaction residues from vMIP-II that reach into the binding pocket and interact with key residues D262, D97, S285, and E288 of CXCR4 in both chemokine recognition sites 1 and 2 (21). ProCA32.CXCR4 was bacterially expressed and purified following our previously reported protocol (20). ProCA32.CXCR4 is also composed of lysine or cysteine residues, which allow post-expression PEGylation (22). PEGylation was verified by Coomassie brilliant blue staining and iodine staining (fig. S1). The CXCR4-targeting capability of ProCA32.CXCR4 was verified and quantified by immunofluorescence staining and enzyme-linked immunosorbent assay (ELISA). We determined the dissociation constant (Kd) of ProCA32.CXCR4-CXCR4 interaction using indirect ELISA (Fig. 2B). Nontargeted ProCA32 was used as a negative control. The binding curve indicated a 1:1 binding stoichiometry, and the determined Kd value was 1.10 0.18 M. The CXCR4 receptor number per Mel290 cell was 1.2 0.1 106. Immunofluorescence staining of ProCA32.CXCR4 after incubating with the CXCR4-expressing cell line Mel290 confirmed that ProCA32.CXCR4 binds to CXCR4 with a high spatial correlation (Pearsons r = 0.82) (Fig. 2C). We hypothesized that intravenous tail injection of ProCA32.CXCR4 would bind to tumors with elevated expression of CXCR4 and enhance the intensity of the corresponding areas in MRI, as demonstrated in Fig. 2D.

(A) Model structure of ProCA32.CXCR4 interacting with CXCR4 [Protein Data Bank (PDB): 4RWS] through targeting moiety. ProCA32.CXCR4 was constructed by engineering the CXCR4 targeting moiety (red) to ProCA32 (blue) by a flexible linker (green). The targeting moiety of ProCA32.CXCR4 binds to CXCR4 through residue-residue and electrostatic interactions. ProCA32.CXCR4 has two Gd3+ (red circle) binding sites. (B) CXCR4 targeting study of ProCA32.CXCR4 by ELISA. The dissociation constant of ProCA32.CXCR4 binding to CXCR4 was calculated as 1.10 0.18 M, measured by indirect ELISA. n = 3. The nontargeted contrast agent ProCA32 did not exhibit CXCR4 targeting capability. (C) Fluorescence staining of Mel290 cells to study the CXCR4 binding effect of ProCA32.CXCR4. Blue fluorescence is nucleus staining with 4,6-diamidino-2-phenylindole (DAPI), green color is fluorescein-labeled ProCA32.CXCR4, red color indicates CXCR4 staining, and composite is the combination of nucleus, CXCR4, and ProCA32.CXCR4 staining. ProCA32.CXCR4 exhibited good spatial colocalization with CXCR4; Pearsons r is 0.82. (D) Working flow of ProCA32.CXCR4. ProCA32.CXCR4 was administered through tail vein injection and distributed with blood flow, and specific targeting to CXCR4 high expression metastatic UM (indicated by black cells) was shown over time.

The r1 and r2 values per Gd3+ for ProCA32.CXCR4 were 30.9 mM1 s1 and 43.2 mM1 s1, respectively, at 1.5 T (Fig. 3A and fig. S2A, relaxivity reported on the basis of per Gd3+ value). Both r1 and r2 relaxivity values were 8 to 10 times greater than the clinically approved Gd3+-based contrast agents (GBCAs) (Fig. 3A and table S1). ProCA32.CXCR4 also exhibited good relaxivities at higher magnetic field of 7.0 T (fig. S2B). The r1 and r2 relaxivity values of non-PEGylated ProCA32.CXCR4 were 23.5 and 98.6 mM1 s1, respectively. The relaxivities of the non-PEGylated form of ProCA32.CXCR4, lysine-PEGylated ProCA32.CXCR4 (Lys-ProCA32.CXCR4), and cysteine-PEGylated ProCA32.CXCR4 (Cys-ProCA32.CXCR4) did not exhibit significant differences. The r1 and r2 relaxivities of ProCA32.CXCR4 were largely retained after PEGylation. Overall, ProCA32.CXCR4 exhibited improved r1 and r2 relaxivities when compared with clinical GBCA at both 1.5 and 7.0 T.

(A) Relaxivity assessment of ProCA32.CXCR4 and GBCA with 60-MHz relaxometer; ProCA32.CXCR4 has 8 to 10 times higher r1 and r2 values than clinical GBCA. (B) Transmetalation study of ProCA32.CXCR4 and other GBCA in the presence of Zn2+. Thermodynamic index of ProCA32.CXCR4 incubated at 37C with Zn2+ was 0.96, which is better than ProHance (0.93) and Gadovist (0.95). (C) Metal (Zn2+, Ca2+, Gd3+, and Tb3+) binding affinity and metal selectivity values of ProCA32.CXCR4 in comparison with clinical contrast agents. N/A, not available; PEG, polyethylene glycol.

Gd3+-related toxicity, such as the development of nephrogenic systemic fibrosis (NSF) in patients with chronic kidney disease and brain deposition of GBCA, is largely attributed to the kinetic and thermodynamic stability of GBCA (23). ProCA32.CXCR4 is stable up to 14 days when incubated with serum at 37C (fig. S3A). The transmetalation study (Fig. 3B) indicated that the ProCA32.CXCR4 complex with Gd3+ has the highest stability in the presence of Zn2+, with a higher thermodynamic index [R1(t) = 4320 min/R0(t) = 0 min] of 0.96, greater than Gadovist (0.95), ProHance (0.93), and Dotarem (0.93) (fig. S3B). Other linear reagents such as Magnevist (gadopentetate) and Eovist (gadoxetate) cannot protect Gd3+ against transmetalation by Zn2+, and relaxivity measurements of those contrast agents were significantly reduced when incubated in the presence of Zn2+ (Fig. 3B and fig. S3B). Using our developed chelator-buffer system method (20), we determined the Gd3+ binding affinity of ProCA32.CXCR4 by competing with preloaded terbium (Tb3+) (fig. S4, A and B). ProCA32.CXCR4 exhibited superior metal selectivity for Gd3+ over Zn2+ [log (KGd/KZn) =16.1] (Fig. 3C and fig. S4C), which was 1011 to 1012 orders of magnitude higher than small chelator contrast agents. For other physiological metal ions such as Ca2+, ProCA32.CXCR4 also exhibited better metal selectivity than small chelator GBCAs, such as Dotarem (gadoterate meglumine) and ProHance (gadoteridol) (Fig. 3C and fig. S4D).

We demonstrated the unique imaging capability of ProCA32.CXCR4 for detection of liver metastases, improving the current detection limit and enabling nodular pattern detection in metastatic M20-09-196 mice at 7.0 T. The early detection of UM metastases in the liver of M20-09-196 mice can be achieved using either Cys-ProCA32.CXCR4 or Lys-ProCA32.CXCR4. Liver micrometastases ranging from 0.01 to 0.08 mm3 were detected with spin echo acquisition and fast spin echo acquisition following tail vein injection of Cys-ProCA32.CXCR4 (0.025 mmol/kg) (Fig. 4A). Enhancement of UM metastases was not detected by MRI following administration of Eovist or Lys-ProCA32 without the targeting moiety (fig. S5A). These results demonstrate the sensitivity and specificity of our system. These small liver lesions, detected by MRI with Cys-ProCA32.CXCR4, were further verified by detailed hematoxylin and eosin (H&E) staining analysis and found to be exclusively nodular growth pattern type (labeled by the yellow, blue, green, and red circles) (Fig. 4B). The interlesion distances and diameters of lesions on MRI correlated well with the corresponding measurements in H&E staining of tissue sections (y = 1.09x + 0.08) (Fig. 4C). A statistical analysis indicated that MRI results can readily differentiate the tumor area from the healthy liver tissue, with an area under the curve (AUC) of 0.84 (Fig. 4D). IHC staining of S100 and CXCR4 further confirmed the lesional areas to be metastatic UM and the CXCR4 expression on metastatic UM (Fig. 4B). Following the same imaging protocol, a mouse model with Lys-ProCA32.CXCR4 injection, in place of Cys-ProCA32.CXCR4, exhibited post-injection enhancement of metastases (fig. S5B).

(A) T1-weighted spin echo and T2-weighted fast spin echo MR images of M20-09-196 before and 48 hours after Cys-ProCA32.CXCR4 injection. At 48 hours after injection, both T1- and T2-weighted MR images revealed four lesions not observed before injection. The zoom-in view of the yellow rectangular region shows both gray and color scales. (B) H&E and IHC staining of M20-09-196 liver with UM metastases. H&E staining revealed four metastatic lesions, highlighted by different color circles, with similar locations as the metastases in MRI images. Higher-magnification images identified the growth pattern of metastases to be nodular pattern. S100 IHC labeling confirmed that the lesions were metastatic UM. CXCR4 immunohistological staining confirmed the CXCR4 expression on UM metastases. (C) The measurement of distances between metastases and the diameter of metastases in MRI images correlates with the H&E histological staining (y = 1.09x + 0.08). (D) Statistical analysis indicated that Cys-ProCA32.CXCR4 provides diagnostic validation for UM metastases in the liver. AUC = 0.84; P < 0.0001. Three mice were used for the experiment. Analyses were based on 11 metastases found on MR images. a.u., arbitrary units.

We further evaluated and validated in vivo the molecular imaging capability of ProCA32.CXCR4 at 4.7 T by generating a liver-implanted UM murine model by inoculation of the Mel290 UM cell line. Molecular dynamic contrast imaging (MDCI) was performed to display implanted UM tumor in mouse liver by administration of Cys-ProCA32.CXCR4 via intravenous injection, followed by the acquisition of T1-weighted gradient echo MRI as a function of time. The nontargeted contrast agent Lys-ProCA32 was used as a control. The tumor regions exhibited different enhancement patterns between mice with Lys-ProCA32 and Cys-ProCA32.CXCR4 injection. In implanted Mel290 mice with Lys-ProCA32 injection, the tumor MRI signal intensity increased at 12 and 50 min after injection and decreased 3 hours after injection (Fig. 5A). However, the tumor MRI signal intensity in the Mel290 mice with Cys-ProCA32.CXCR4 injection gradually increased to the maximum at 22 hours after injection and then began to decrease due to excretion (Fig. 5A). The time plot of UM tumor signal-to-noise ratio (SNR) change followed by Cys-ProCA32.CXCR4 injection showed that UM tumor SNR increased more than 40% at 22 hours after injection when compared with before injection, whereas SNR of tumor region in the Mel290 mice with Lys-ProCA32 injection showed a mild increase (10%) immediately after injection (12 min) and then washed out at 3 hours after injection (Fig. 5B). On the other hand, MRI results of Mel290 mice with Cys-ProCA32.CXCR4 and Lys-ProCA32 injection exhibited similar patterns of SNR changes in the liver regions over time (Fig. 5C). The liver SNR of both mice with Cys-ProCA32.CXCR4 and Lys-ProCA32 injection increased drastically right after injection and up to 3 hours, with a percentage increase of SNR of approximately 45% at 3 hours after injection when compared with before injection. This enhancement of the liver region gradually decreased due to elimination. Cys-ProCA32.CXCR4 was observed to target and distribute across the tumor tissue in Mel290 mice. The MRI of tumor regions in Mel290 mice following Cys-ProCA32.CXCR4 injection revealed enhancement of the tumor rim immediately after injection and rapid penetration into the center (Fig. 5, A and D). The immunofluorescence staining of the administered Cys-ProCA32.CXCR4 into Mel290 murine tumor tissue exhibited intense and broadly distributed red immunofluorescence labeling (Fig. 5E). In contrast, red fluorescence staining was not observed with the tumor tissues of the Mel290 mice receiving the Lys-ProCA32 injection. Gd3+ content analysis using inductively coupled plasma optical emission spectrometry (ICP-OES) indicated that the tumor tissue of Mel290 mice receiving the Cys-ProCA32.CXCR4 injection exhibited significantly higher Gd3+ content than tumor tissue of Mel290 mice with the Lys-ProCA32 injection (Fig. 5F). These results further validated the CXCR4-targeting capability of ProCA32.CXCR4 in vivo with good tumor permeability.

(A) T1-weighted gradient echo MR images of control mice (with injection of nontargeted agent Lys-ProCA32) and mice with Cys-ProCA32.CXCR4 injection. MRI scans were acquired before and after injection at different time points until 46 hours; tumors are represented by the heat map in MRI images. (B) Percentage increase of SNR of melanoma tumors at different time points shows the dynamic binding process of Cys-ProCA32.CXCR4. For mice that received the Cys-ProCA32.CXCR4 injection, a gradual increase of intensity in melanoma tumor region was observed up to 24 hours, showing the CXCR4-targeting effect, followed by washing out at 46 hours (further time points not acquired). (C) Time plot of the liver SNR percentage increase following Cys-ProCA32.CXCR4 and Lys-ProCA32 injection. The liver SNRs of mice receiving Cys-ProCA32.CXCR4 and Lys-ProCA32 exhibited similar patterns of the SNR time plots, where the liver intensity substantially increased up to 3 hours after injection of both contrast agents, followed by loss of intensity after 3 hours. (D) Time plot of tumor rim and tumor center SNR change of mice with Cys-ProCA32.CXCR4 administration. Cys-ProCA32.CXCR4 exhibited good tumor permeability; tumor rim SNR was enhanced at early time points (12 min after injection). SNR enhancement gradually penetrated to the center of the tumor. At 24 hours after injection, the view of the tumor region following Cys-ProCA32.CXCR4 injection revealed broad distribution and heterogeneous enhancement. (E) Immunofluorescence staining of Cys-ProCA32.CXCR4 and Lys-ProCA32 in the liver (L) and tumor (T) of Mel290 mice. For mice that received Cys-ProCA32.CXCR4 injection (top), Cys-ProCA32.CXCR4 accumulated in the UM tumor tissue (denoted by red fluorescence). For the mice injected with Lys-ProCA32 (bottom), UM tumors exhibited dark fluorescence intensity relative to the UM tumor regions of the mice that received Cys-ProCA32.CXCR4 injection. (F) ICP-OES analysis of Gd3+ tissue distribution 2 days after injection of ProCAs. Mice with Cys-ProCA32.CXCR4 injection exhibited significantly more Gd3+ distribution in tumor tissue than mice that received Lys-ProCA32 injection (P < 0.01).

We validated the in vivo CXCR4 targeting capability of Cys-ProCA32.CXCR4 by receptor blocking experiment. A subcutaneous UM murine model was developed to demonstrate that UM tumor signal intensity enhancement following Cys-ProCA32.CXCR4 administration could be blocked by first administering the CXCR4 blocking reagent (Fig. 6A). We specifically constructed a CXCR4 blocking reagent by fusing the CXCR4-targeting moiety (LGASWHRPDKFCLGYQKRPLP) of ProCA32.CXCR4 to the C terminus of glutathione S-transferase (GST) tag to ensure proper blocking. Injection of the nontargeted Lys-ProCA32 only resulted in initial SNR enhancement at 3 hours after administration due to blood pool distribution. This enhancement returned to baseline at 24 hours. In contrast, injection of Cys-ProCA32.CXCR4 resulted in maximum SNR enhancement at 24 hours after injection and returned to the baseline at 48 hours. Previous injection of CXCR4 receptor blocking reagent specifically eliminated the enhancement at 24 hours by Cys-ProCA32.CXCR4 but retained the 3-hour initial enhancement due to blood pool effect (Fig. 6, B and C, and fig. S6). These results support the view that ProCA32.CXCR4 is able to specifically bind to the CXCR4 receptor overexpressed on the tumors and enables molecular targeting MRI.

(A) Comparison of subcutaneous UM tumor intensity change on T1-weighted MRI images following administration of Cys-ProCA32.CXCR4 with and without previous administration of blocking reagent; subcutaneous UM tumors are represented by color heat map. Tumor from UM mice that received Cys-ProCA32.CXCR4 injection showed significant increase in MRI signal intensity after Cys-ProCA32.CXCR4 administration. This enhancement could be blocked by first administrating the CXCR4 receptor blocking reagent. (B) Comparison of UM tumor SNR change following administration of Cys-ProCA32.CXCR4 and blocking reagent + Cys-ProCA32.CXCR4. For the mice that received Cys-ProCA32.CXCR4 injection, the SNR of UM tumor substantially increased at 24 hours after administration. This enhancement was blocked by first administrating a blocking reagent. As seen with the mice that received the blocking reagent and then the Cys-ProCA32.CXCR4 injection, the SNR of UM tumor was notably lower in comparison with the UM tumor SNR of the mice with Cys-ProCA32.CXCR4 administration. (C) UM tumor SNR change following administration of Cys-ProCA32.CXCR4, blocking reagent + Cys-ProCA32.CXCR4, and Lys-ProCA32. At 3 hours after administration, mice from all three groups showed an SNR increase. At 24 hours, mice with blocking reagent + Cys-ProCA32.CXCR4 administration and mice with Lys-ProCA32 administration showed SNR washout, while mice that received Cys-ProCA32.CXCR4 exhibited further SNR increases compared with 3 hours. At 48 hours, mice that received blocking reagent + Cys-ProCA32.CXCR4 and mice that received Lys-ProCA32 administration exhibited a further SNR decrease. Mice that received Cys-ProCA32.CXCR4 administration showed SNR washout at 48 hours in comparison with 24 hours.

A detailed pharmacokinetic study was carried out to study the bioavailability of Cys-ProCA32.CXCR4. The AUC0-72h (fig. S7A) of ProCA32.CXCR4 was 113.20 gh/ml. The clearance of ProCA32.CXCR4 was 0.31 ml/min per kilogram, slightly less than Eovist (0.4 ml/min per kilogram). ProCA32.CXCR4 had a half-time of 9.19 hours, with a mean residence time of 19.58 hours. The biodistribution study using ICP-OES showed very low amounts of Gd3+ in the brain [0.07% injection dosage (ID)/g tissue] at 5 days after injection of ProCA32.CXCR4 (fig. S7), with the liver displaying the highest concentration of Gd3+ (21.3% ID/g tissue). The biodistribution studies of Gd3+ demonstrated no potential Gd3+-dependent toxicity via brain deposition. Alkaline phosphatase (ALP) and Alanine transaminase (ALT) levels of the mice injected with ProCA32.CXCR4 were comparable with levels from control mice. Albumin, total bilirubin, bilirubin-conjugated, and bilirubin-unconjugated levels in mice injected with ProCA32.CXCR4 exhibited no substantial differences when compared with control mice (table S2). Detailed histological analyses of brain, liver, spleen, muscle, and kidney tissues showed no observable tissue damage (fig. S8). Thus, injection of ProCA32.CXCR4 did not indicate acute toxicity in the mouse study.

The liver is a common site for cancer metastases. UM almost exclusively metastasizes to the liver. The mechanism of the liver-specific metastases is not well understood. One of the hypotheses in the field postulates that tumor cells that overly express CXCR4 hijack the CXCR4/CXCL12 axis during the metastatic process and spread to the liver (1315). This hypothesis is based on the findings that the liver microenvironment in UM is rich in multiple chemoattractants including CXCL12, the natural ligand of CXCR4 (24), and CXCR4 was found to be overexpressed on UM cells in several UM cell line studies (17, 25). CXCR4 is proposed to be a prognostic marker in multiple malignancies, including acute myelogenous leukemia, breast cancers, colorectal cancers, and cutaneous melanoma (17, 2628). Thus, development of imaging agents for CXCR4 may be used as a diagnostic biomarker in cancer and potentially as a prognostic factor.

In this investigation, we validated the diagnostic value of CXCR4 as an imaging biomarker in UM by demonstrating elevated CXCR4 expression in three different biological systems: UM patient liver metastases, UM cell lines, and an in vivo UM murine model. Multiple attempts have been made toward the development of CXCR4 molecular imaging agents over the years using different imaging technologies including Single-photon emission computed tomography (SPECT), PET, and near-infrared imaging (2931). MRI has the advantage of being able to provide high spatial resolution imaging without ionizing radiation and depth limitation. Despite this advantage, the application of MRI in molecular imaging is very challenging due to the sensitivity of current contrast agents and the low concentration of biomedical receptors presented on the tumor cell surface (32). To overcome these challenges, we developed a CXCR4-targeted MRI contrast agent, ProCA32.CXCR4, which exhibits 8- to 10-fold increases in both r1 and r2 relaxivities over clinical GBCA and enables sensitive MRI detection of CXCR4. We generated a metastatic UM mouse model by inoculation of M20-09-196 melanoma cells to demonstrate the imaging capacity of ProCA32.CXCR4. MRI following ProCA32.CXCR4 administration is able to detect UM micrometastases (Fig. 4A and fig. S5B) as small as 0.1 mm3 in murine livers, which is a notable improvement in the detection limit of MRI for liver lesions (10). Several factors contributed to the robust detection of micrometastases at early stages. First, CXCR4 targeting enabled ProCA32.CXCR4 accumulation at metastasis sites. Second, the high relaxivities of ProCA32.CXCR4 substantially improved the sensitivity of MRI. ProCA32.CXCR4 has a secondary coordination shell and optimized rotational correlation time, which contributes to the improvement in relaxivities compared with small molecule chelators (33). Furthermore, both high r1 and r2 of ProCA32.CXCR4 increased the confidence in the system and avoided artifacts of detection by applying both T1- and T2-weighted acquisition. Another challenge in imaging UM metastases in the liver is to identify pathological growth patterns of metastases with MRI (11). MR images of M20-09-196 mice following administration of ProCA32.CXCR4 exclusively enhanced nodular growth pattern metastases, and this may provide an approach to identify nodular growth pattern lesions with MRI.

Administration of ProCA32.CXCR4 achieved detection of liver metastases using MDCI by MRI. In the Mel290 murine model, intensity changes over time exhibited different patterns in UM than in adjacent liver tissue (Fig. 5, B and C). Tumor region intensity steadily increased up to 24 hours after Cys-ProCA32.CXCR4 injection due to in vivo dynamic binding to CXCR4, followed by slow washout after 24 to 48 hours. We measured a transient increase (at 12 min) immediately after Lys-ProCA32 injection due to in vivo distribution, with subsequent washout after 3 hours. Similar enhancement patterns were observed in the liver regions of mice injected with both Cys-ProCA32.CXCR4 and Lys-ProCA32. This special property of ProCA32.CXCR4 provides a possibility of acquiring MDCI using MRI. MDCI provides an additional avenue to noninvasively differentiate tumors from healthy livers by taking advantage of biomarker binding capabilities. In this study, we also demonstrated that ProCA32.CXCR4 exhibits excellent tumor permeability, which is very different from most nanoparticles or chelator-based targeting contrast agents that mostly enhance the tumor boundary (34). This property allows the mapping of heterogeneous CXCR4 expression inside the tumor (Fig. 5, A and D) and may facilitate monitoring of changes in CXCR4 expression through the tumor tissue during progression and treatment.

Since the FDA approval of gadopentetate dimeglumine (Magnevist; Bayer HealthCare Pharmaceuticals) in 1988, GBCAs have been widely used for clinical MRI imaging. However, the potential for NSF and Gd3+ brain deposition in patients and animals has raised concerns over the use of GBCAs (3537). We have carefully considered these factors in the design of ProCA32.CXCR4 for translation into the clinic. The Gd3+ binding sites of ProCA32.CXCR4 were designed to balance Gd3+ binding for safety and water accessibility for relaxivities. ProCA32.CXCR4 has been shown to exhibit unprecedented Gd3+ kinetic and thermodynamic stability, with a log (KGd) of ProCA32.CXCR4 calculated at 21.89. Metal selectivity values of ProCA32.CXCR4 for Gd3+ over Zn2+ and Ca2+ are 106 to 1012 times greater than the clinically approved contrast agents Dotarem and ProHance. The inertness of ProCA32.CXCR4 in the presence of Zn2+ verified its strong stability against transmetalation. Moreover, the improved relaxivity of ProCA32.CXCR4 enabled excellent contrast enhancement in vivo with 75% reduction of Gd3+ dosage compared with other GBCAs. Acute toxicity and tissue/organ toxicity in the in vivo model were not observed. Collectively, ProCA32.CXCR4 has a safe profile, which includes strong Gd3+ binding affinity, unique metal selectivity, and inertness against transmetalation. In addition, no acute toxicity and/or tissue/organ toxicity was observed in the in vivo model. These results strongly support the safety of ProCA32.CXCR4 for diagnostic use due to the observed strong Gd3+ binding affinity, unique metal selectivity, and inertness against transmetalation.

We acknowledge potential limitations in the translatability of our system, as images with best tumor enhancement happened between 24 and 48 hours after injection. We are in the process of optimizing polyethylene glycol (PEG) modification of ProCA32.CXCR4 to tune the pharmacokinetic/pharmacodynamic (PK/PD) properties. We are aware that CXCR4 can be expressed on normal cells (i.e., immune cells). Further studies will be conducted to more extensively evaluate ProCA32.CXCR4 before it can be considered for clinical applications.

The present research validates our hypothesis that CXCR4 may be a diagnostic imaging biomarker for liver metastases. These results were measured using UM patients samples, UM cell lines, and animal models. In addition, we successfully designed a CXCR4-targeting protein-based contrast agent, ProCA32.CXCR4, for early detection of UM hepatic metastases. The detected liver micrometastases were validated by histological analyses and correlated with MRI results. Collectively, our results indicate that this contrast agent can enable precision MRI capable of defining molecular signatures for identifying metastases and possibly for treatment stratification.

Metastatic liver tissue from UM patients was immunolabeled with anti-CXCR4 antibodies for IHC analyses. Briefly, liver tissue was fixed in 10% neutral-buffered formalin and embedded in paraffin blocks. These blocks were sectioned at a thickness of 5 m for the labeling. Paraffin-embedded sections were first deparaffinized and rehydrated following a mixture of one part of 30% hydrogen peroxide and nine parts of absolute methanol to quench endogenous peroxidase activity for 10 min. Samples were then washed three times using tris-buffered saline with Tween 20 (TBST), 5 min each wash. Antigen retrieval was achieved by boiling in target retrieval solution (Agilent Technologies) for 20 min. Slides were washed as before prior to blocking in a 5% bovine serum albumin (BSA; Thermo Fisher Scientific) in TBST for 2 hours. Samples were incubated in a 1:300 dilution of the anti-CXCR4 primary antibody (Abcam, 12G5, ab189048) in TBST overnight at 4C. IHC staining was performed with a red chromogen kit following the manufacturers guidelines. Counterstaining of the nucleus was performed with hematoxylin. A CXCR4-positive control (brain specimen) was processed with the same protocol. All cases of UM hepatic metastases exhibited high expression of CXCR4, indicated by the red-labeling intensity.

Flow cytometry was performed to measure the percentage of CXCR4+ UM cells. Cultured human UM cells were dissociated with a non-enzymatic cell dissociation solution (Sigma-Aldrich, St. Louis, MO), washed, and immunolabeled for 20 min at 4C with an allophycocyanin (APC) mouse anti-human CD184 antibody (CXCR4 is also known as CD184, clone 12G5) (BD Biosciences, San Jose, CA). Data acquisition was performed using a BD FACSAria IIu cell sorter (BD Biosciences, San Jose, CA). FlowJo software (Tree Star, Ashland, OR) was used for data analysis.

ProCA32.CXCR4 was constructed by engineering a CXCR4-targeting moiety (LGASWHRPDKFCLGYQKRPLP) to the C terminus of ProCA32; PEGylation was performed for surface modification. ProCA32.CXCR4 was expressed in BL21 (DE3) pLysS cell strain and purified following our established protocol (20). Two site-specific PEGylations, cysteine PEGylation and lysine PEGylation, were used for ProCA32.CXCR4 surface modification. For cysteine PEGylation, ProCA32.CXCR4 solution [concentration between 1 and 10 mg/ml, in 10 mM HEPES (pH 7.2)] was degassed by bubbling with nitrogen. Tris (2-carboxyethyl) phosphine hydrochloride (Sigma-Aldrich) solution was used to reduce disulfide bonds at room temperature for 20 min. Methoxy PEG maleimide (JenKem Technology) with a molecular weight of 2 kDa was reacted with reduced ProCA32.CXCR4 at a molar ratio of 1:1 overnight at 4C. For lysine PEGylation, ProCA32/ProCA32.CXCR4 solution [concentration between 1 and 10 mg/ml, in 10 mM HEPES (pH 7.2)] was reacted with methoxy PEG succinimidyl carboxymethyl ester reagent (molecular weight of 2 kDa, JenKem Technology) at a molar ratio of 1:5 overnight at 4C. Purification of the PEGylated protein sample was achieved by fast protein liquid chromatography. The PEGylation product was evaluated with Coomassie blue staining and iodine (I2) staining (fig. S1).

The relaxation times (T1 and T2) of ProCA32.CXCR4 were measured with 1.5 T Bruker minispec relaxometer and 7.0 T Bruker MRI scanner. We tested different concentrations of ProCA32.CXCR4 and GdCl3 (1:2) prepared in a solution of 50 mM HEPES, 100 mM NaCl, with a pH of 7.2. Samples were incubated at 37C for 1 hour before measurement. T1 and T2 relaxation times of ProCA32.CXCR4 at 1.5 T were measured by a 1.5 T Bruker minispec relaxometer, and longitudinal (r1) and transverse (r2) relaxivities were calculated in Eq. 1. The slopes of curves were the r1 and r2 relaxivities (fig. S2A). Relaxivities of ProCA32.CXCR4 at 7.0 T were measured with a 7.0 T Bruker MRI scanner with saturation recovery and spin echo sequence (fig. S2B). Commercially available GBCAs (i.e., Dotarem, Magnevist, and Eovist) were prepared in the same buffer and measured using the same procedures.ri=1Tsample1Tbuffer[Gd3+]T(1)

Immunofluorescence staining of CXCR4 was performed on cultured Mel290 and M20-09-196 UM cells. Cultured cells were harvested upon reaching 50 to 70% confluency and fixed on cover slides with 3.7% formaldehyde solution at 4C. Fixed cells were incubated with 5 M fluorescein 5-carbamoylmethylthiopropanoic acid N-hydroxysuccinimide ester-labeled ProCA32.CXCR4 (the control group was incubated with fluorescein-labeled ProCA32) for 1 hour at 37C. Briefly, the slides were washed thoroughly with TBST buffer, and then the nucleus was labeled using 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific) and imaged with a Zeiss microscope. For the colocalization studies, cells were incubated with fluorescein-labeled ProCA32.CXCR4 followed by blocking with 5% BSA (prepared in TBST buffer) for 20 min at room temperature and overnight incubation with 0.1% dilution of anti-CXCR4 (Abcam, ab189048) at 4C. UM cells were washed and incubated with a 0.1% dilution of a goat anti-mouse secondary antibody (Invitrogen, Alexa Fluor 555) for 60 min at room temperature. DAPI (Thermo Fisher Scientific) was applied for nuclear labeling before slides were covered and sealed. Colocalization analyses of red fluorescence (555-nm excitation) and green fluorescence (488-nm excitation) were done using Fijis plugin coloc2 (Fig. 2C).

Flash-frozen liver tissues of Mel290-inoculated mice were collected after injection of either ProCA32.CXCR4 or ProCA32 (control group) (Fig. 5E). Liver cryosections (4 m) were thawed at room temperature for 20 min and rehydrated with TBST. Tissue sections were surrounded with a hydrophobic barrier using Dako pen (Agilent) and blocked with 5% BSA for 60 min at room temperature, followed by incubation with an anti-ProCA32.CXCR4 or anti-ProCA32 primary antibody (1:50 dilution) for 60 min at room temperature. After thoroughly washing with TBST, tissue slides were incubated with 0.1% dilution of goat anti-rabbit secondary antibody (Invitrogen, Alexa Fluro 555) for 60 min at room temperature. Nuclear labeling proceeded by using DAPI (Thermo Fisher); slides were covered and sealed.

An indirect ELISA assay was used to quantify the CXCR4-targeting capability. Cell lysates of Mel290 cells in NaHCO3 solution (pH 9.6) were incubated in 96-well plates overnight at 4C. The 96-well plates were washed thoroughly in TBST buffer and blocked by 5% BSA solution (prepared in TBST) for 60 min at room temperature. Different concentrations of ProCA32.CXCR4, ranging from 0 to 5000 nM prepared in TBST, were added and incubated for 1 hour at room temperature. A 0.1% solution with an anti-ProCA32.CXCR4 antibody (in-house polyclonal rabbit antibody) was added for 60-min incubation at room temperature. As a secondary antibody, we used a stabilized goat anti-rabbit horseradish peroxidaseconjugated antibody (Pierce) for 45 min at room temperature. After washing with TBST, 100 l of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added into each well to visualize the color change. When a blue gradient color was observed, 100 l of 1 M H2SO4 was added into each well to stop the reaction. The absorbance intensity at 450-nm wavelength was measured by a FLUOstar OPTIMA plate reader, and data were plotted using GraphPad Prism 5.

The Gd3+ binding affinity of ProCA32.CXCR4 was investigated by a Tb3+ competition assay in a chelator buffer system (20). QM1 fluorescence spectrophotometer (PTI) was used to collect fluorescence spectra at room temperature. A Tb3+ luminescence resonance energy transfer (LRET) experiment was used to determine the Tb3+ binding affinity of ProCA32.CXCR4. Tb3+ LRET emission spectra were recorded from 500 to 600 nm wavelength with tryptophan excitation at 280 nm (fig. S4A). The chelator buffer system consisted of 30 M ProCA32.CXCR4, 5 mM diethylenetriamine pentaacetic acid (DTPA), 50 mM HEPES, and 150 mM NaCl (pH 7.2). DTPA is a strong chelator (Kd = 1021 M, 25C, National Institute of Standards and Technology). Upon titration of different concentrations of Tb3+ titrated into the solution, the free Tb3+ concentration can be calculated by[Tb3+]free=KdTb,DTPA[TbDTPA][DTPA]free(2)where [Tb-DTPA] is the Tb3+ -DTPA complex concentration, and it is assumed that the free Tb3+ triggered the terbium-tryptophan LRET and caused fluorescence signal change. The Tb3+ binding affinity to ProCA32.CXCR4 was determined byf=[Tb3+]freenKdTb,ProCA32.CXCR4n+[Tb3+]freen(3)where f is the fractional LRET signal change and n is the Hill number.

Gd3+ binding affinity of ProCA32.CXCR4 was determined by competing with Tb3+-loaded ProCA32.CXCR4. ProCA32.CXCR4 (10 M) and Tb3+ (20 M) were prepared in 5 mM DTPA, 50 mM HEPES, and 150 mM NaCl at pH 7.2. Different concentrations of Gd3+, ranging from 0 to 200 M, were added and incubated overnight. Gd3+ replacement of Tb3+ in the ProCA32.CXCR4 binding pockets resulted in a signal decrease in fluorescence spectra (fig. S4B), and an apparent Kd of Gd3+ competition was calculated byf=([Tb]T+[Gd]T+Kdapp)([Tb]T+[Gd]T+Kdapp)24[Tb]T[Gd]T2[Tb]T(4)where f is the fractional LRET signal change, [Tb]T is the total Tb3+ concentration, [Gd]T is the total Gd3+ concentration, and Kdapp is the apparent dissociation constant of Gd3+ in competition with Tb3+.

The dissociation constant of Gd3+ with ProCA32.CXCR4 was then calculated byKdGd,ProCA32.CXCR4=KdappKdTb,ProCA32.CXCR4KdTb,ProCA32.CXCR4+[Tb3+]T(5)where KdTb,ProCA32.CXCR4 is the dissociation constant of Tb3+ with ProCA32.CXCR4 and [Tb3+]T is the total Tb3+ concentration.

The dissociation constant between ProCA32.CXCR4 and Ca2+ was determined in an EGTA buffer system. ProCA32.CXCR4 (10 M) was prepared in EGTA buffer [5 mM EGTA, 50 mM HEPES, and 150 mM NaCl (pH 7.2)]. Free Ca2+ concentration was calculated by Tsiens assay (38), using Eq. 6[Ca2+]free=KdCa,EGTA[Ca2+EGTA][EGTA]free(6)

The tryptophan fluorescence change triggered by the increase of the free calcium (fig. S4D), Kd of Ca2+ binding to ProCA32.CXCR4, can be fit byf=[Ca2+]freenKdCa,ProCA32.CXCR4n+[Ca2+]freen(7)where f is the fractional fluorescence change, [Ca2+]free is the free Ca2+ concentration, and n is the Hill number.

The dissociation constant between Zn2+ and ProCA32.CXCR4 was determined by a modified fluorescence competition assay, where 2 M ZnCl2 and FluoZin-1 (Thermo Fisher) were combined in a 1:1 ratio. Different concentrations of ProCA32.CXCR4 were titrated into the sample, and fluorescence emission spectra were recorded from 500 to 600 nm following excitation at 495-nm wavelength (fig. S4C). The apparent Kd of ProCA32.CXCR4 -Zn2+ competition was calculated byf=([Zn]T+[ProCA32.CXCR4]T+Kdapp)([Zn]T+[ProCA32.CXCR4]T+Kdapp)24[Zn]T[ProCA32.CXCR4]T2[Zn]T(8)where [Zn]T is the total Zn2+ concentration, [ProCA.CXCR4]T is the total ProCA32.CXCR4 concentration, and Kdapp is the apparent dissociation constant. Using the known dissociation constant of Zn2+ to Fluozin-1, the dissociation constant between Zn2+ and ProCA32.CXCR4 was calculated using Eq. 9KdZn,ProCA32.CXCR4=KdappKdZn,FluozinKdZn,Fluozin+[Fluozin]T(9)where Kdapp is the apparent dissociation constant of ProCA32.CXCR4 and Zn2+ in competition with Fluozin-1.

To characterize the resistance of the Gd3+-ProCA32.CXCR4 complex to transmetalation by endogenous ions such as Zn2+, a relaxometric transmetalation assay was performed using a previously reported test by Laurent and colleagues (39). Briefly, ProCA32.CXCR4 and other GBCA were mixed with the same concentration of ZnCl2 chloride (2.5 mM) in pH 7 phosphate buffer. The final mixture contained 0.026 M KH2PO4, 0.041 M Na2HPO4, 2.5 mM Gd3+ complex, and 2.5 mM ZnCl2. When transmetalation of Gd3+ by Zn2+ occurs, insoluble GdPO4 formed and a decreased proton relaxation rate was observed. The longitudinal relaxation rate change of the mixture reflected the transmetalation process of Gd3+ by Zn2+, and the thermodynamic index was calculated by r1 of the mixture after incubation in the presence of Zn2+ over the initial r1 of GBCA (fig. S3B).

A volume of 150 l from a 500 M ProCA32.CXCR4 was combined with 150 l of mouse serum and incubated at 37C to study serum stability. A total of 15 l of each sample was taken after 3 hours, 4 hours, and 1, 2, 4, 6, 8, and 14 days of incubation. Samples were boiled for 10 min after being mixed with 2 l of 1 M EGTA solution and SDS buffer and analyzed by Ponceau S assay (fig. S3A).

All animal experiments performed in this study complied with Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and complied with an animal protocol reviewed and approved by the Institutional Animal Care and Use Committee at Georgia State University and University of Georgia.

For intraocular melanoma mouse model with hepatic metastases, human UM M20-09-196 cells were inoculated into 10-week-old female NU/NU mice (The Jackson Laboratory, Bar Harbor, ME) to generate the intraocular melanoma mouse model. Aliquots of 106 UM cells were suspended in 2.5 l of phosphate-buffered saline (PBS) buffer and then inoculated into the suprachoroid space of the right eye of each nude mouse using a transcorneal technique. The mice were anesthetized with intraperitoneal injection of ketamine and xylazine mixture. A tunnel was prepared from the limbus within the cornea, sclera, and ciliary body to the choroid with a 301/2-gauge needle under a surgical microscope. The tip of a 10-l glass syringe with a 31-gauge/45 point metal needle (Hamilton, Reno, NV) was used to introduce the cell suspension into the suprachoroid space through the needle track. The eyes were enucleated after 2 weeks of inoculation.

Ten-week-old female NU/NU mice (The Jackson Laboratory, Bar Harbor, ME) were used to establish the intrahepatic heterotopic xenotransplantation tumor model. Mel290 cells were cultured and resuspended in sterile PBS buffer. The mice were anesthetized with a ketamine and xylazine mixture and placed in a supine position. A small incision was made along the right flank of the mouse. The liver was exposed with a small retractor. A surgical microscope was used to guide a 301/2-gauge needle into the liver until its point reached just below the liver subcapsule. Two million Mel290 cells were inoculated in a volume of 20 l of PBS. The needle was then carefully removed at the same time that a sterile swab held to the injection site. The formation of white cell bulla between hepatic parenchyma and the capsule was the criterion for a successful injection. The incision was sutured with a 5-0 absorbable suture. After 2 to 3 weeks following Mel290 cell injection, melanoma tumors formed in the liver.

Ten-week-old female NU/NU mice were used for the subcutaneous UM murine model. Aliquots of 2 106 Mel290 cells were suspended in 50 l of PBS buffer mixed with 50 l of Matrigel Matrix (Corning Life Science) and injected subcutaneously on both the right and left side of the back of NU/NU mice. After 6 weeks, subcutaneous tumors of 60 to 120 mm3 in volume were formed.

M20-09-196 mice were scanned with a 7.0 T Agilent MRI scanner at University of Georgia. Mice were anesthetized by inhalation of isoflurane gas. The respiration rates of animals were monitored throughout the MRI scanning and controlled at 70 to 80 times per minute. T1- and T2-weighted images were collected by spin echo and fast spin echo sequence before and after one bolus injection of Lys-ProCA32.CXCR4 or Cys-ProCA32.CXCR4 (0.025 mmol/kg) at 3, 24, and 48 hours. Control mice were injected with one bolus at the same dosage of Lys-ProCA32 and imaged at the same time points with the same parameters. The parameters of spin echo sequence were as follows: repetition time (TR), 500 ms; echo time (TE), 14.89 ms; field of view (FOV), 3.5 cm 3.5 cm by a matrix of 512 512; thickness, 1 mm with no gap. The parameters of fast spin echo sequence were as follows: TR/echo spacing (ESP), 4000 ms/ 10 ms; FOV, 3.5 cm 3.5 cm by a matrix of 512 512; thickness, 1 mm with no gap.

Intrahepatic xenotransplantation Mel290 mice MR images were all collected on a 4.7-T small-bore Varian MRI scanner at Emory University. Mice were anesthetized following similar procedure, and T1-weighted images were collected before and after one bolus injection of Cys-ProCA32.CXCR4 (0.025 mmol/kg) at 12 min, 50 min, and 3, 22, and 46 hours by gradient echo sequence. The parameters of gradient echo sequence were as follows: TR/TE, 140 ms/11 ms; FOV, 4 cm 4 cm by a matrix of 512 512.

Subcutaneous Mel290 tumor mice MRI results were acquired with a 7.0 T Bruker MRI scanner at Yerkes National Primate Research Center. Mice were anesthetized following a similar procedure as detailed above, and T1-weighted images were collected before and after one bolus injection of Cys-ProCA32.CXCR4 or Lys-ProCA32 (0.025 mmol/kg) at 3, 24, and 48 hours. Blocking group mice received intravenous injections of CXCR4 blocking reagent (0.025 mmol/kg) 24 and 12 hours before the injection of Cys-ProCA32.CXCR4. The parameters of the rapid imaging with refocused echoes (RARE) sequence were as follows: TR/TE, 560 ms/11 ms; FOV, 3.5 cm 3.5 cm by a matrix of 256 256. MRI data were processed and analyzed by Fiji and MRIcron.

ICP-OES was used to analyze the Gd3+ distribution in different mouse organs after injection of ProCA32.CXCR4. Healthy CD-1 mice were injected with a bolus dosage of ProCA32.CXCR4 (0.025 mmol/kg). Animals were euthanized 46 hours after receiving an injection of ProCA32.CXCR4, and heart, liver, spleen, kidney, brain, and muscle tissues were subsequently collected and used for ICP-OES analysis. Tissues (0.1 to 0.5 g) were dissolved overnight in 1 ml of Nitric Acid 67-69%, Optima (Fisher Chemical). Undissolved particles were removed by filtration, and the supernatant was retained for Gd3+ content analysis by ICP-OES (fig. S7B).

ProCA32.CXCR4 acute toxicity was tested by a bolus injection of 100 l of 7 mM ProCA32.CXCR4 to 10-week-old healthy CD-1 mice. ProCA32.CXCR4 solutions with two different PEGylation methods (Cys-ProCA32.CXCR4 and Lys-ProCA32.CXCR4) were tested. Each test group had three mice, and the control group was injected with saline. Mice were observed every 8 hours after injection and then euthanized after 5 days. Terminal blood was collected by cardiac puncture, and serum was transferred immediately to microcentrifuge tube. Plasma was separated from blood cells by centrifugation at 14,000 rpm, 4C for 10 min. Serum samples were used for basic blood biochemistry tests and kidney function tests to measure ALT, ALP, and electrolyte levels (table S2). Tissues including heart, muscle, liver, spleen, kidney, lung, and brain were collected for analysis of gadolinium distribution using ICP-OES.

Female CD-1 mice (8 to 10 weeks old) were used to determine the pharmacokinetic parameters of Cys-ProCA32.CXCR4. Cys-ProCA32.CXCR4 (100 l, 0.025 mmol/kg) was administered through tail vein injection. Blood samples were collected at various time points using the saphenous vein up to 7 days using a sparse sampling design (three to six animals per time point). Immediately following blood sample collection, samples were stored on ice, serum was obtained through centrifugation, and Gd3+ concentration was determined using ICP-OES. Pharmacokinetic parameters were calculated using the noncompartmental analysis tool of Phoenix WinNonlin software. The areas under the concentration-time curve (AUClast and AUCinf) were calculated using a linear trapezoidal rule. The clearance and volume of distribution (Vss) were estimated following intravenous dose administration. The elimination rate constant value (k) was obtained by linear regression of the log-linear terminal phase of the concentration-time profile using at least three declining concentrations in terminal phase with a correlation coefficient of >0.8. The terminal half-life value (T1/2) was calculated using the equation 0.693/k.

SNR was calculated by the mean value across different slides of MRI results of the same subjects. Analyses of differences between the two groups were performed using two-tailed Students t test in GraphPad Prism 5 (GraphPad Software). The P values are denoted in figure legends, and differences were considered significant if P < 0.05. No estimation of sample size and blinding was performed for animal studies. Receiver operating characteristic (ROC) analyses were performed using R and SAS. AUC was reported to measure the performance of the contrast agent. Mice were randomly assigned to groups for the experiments.

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaav7504/DC1

Fig. S1. PEGylation SDS-PAGE gel of protein contrast agents.

Fig. S2. Determination the relaxivity values of ProCA32.CXCR4.

Fig. S3. Serum stability and transmetalation study of ProCA32.CXCR4.

Fig. S4. Determination of ProCA32.CXCR4 metal binding affinities.

Fig. S5. MRI images of metastatic UM mice M20-09-196 before and after administration of Lys-ProCA32, Eovist, and Lys-ProCA32.CXCR4 (n = 2 for Eovist group, n = 3 for Lys-ProCA32 and Lys-ProCA32.CXCR4 group).

Fig. S6. T1-weighted MRI images of subcutaneous UM mice before and after administration of Cys-ProCA32.CXCR4, blocking reagent + Cys-ProCA32.CXCR4, and Lys-ProCA32 (n = 3 for each group).

Fig. S7. Pharmacokinetic study of Cys-ProCA32.CXCR4 and ICP-OES analysis of Gd3+ content in different mouse organs.

Fig. S8. H&E staining analysis of mice tissues collected 7 and 14 days after injection of Cys-ProCA32.CXCR4.

Table S1. Relaxivities of investigated contrast agents in 10 mM Hepes at 37C.

Table S2. Clinical pathology profile of mouse serum.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank R. C. Long for operating the 4.7 T small-animal MRI scanner. We thank S. E. Woodman, T. A. McCannel, and B. L. Burgess for providing cell lines. We thank M. Kirberger for critical review and editing of this manuscript. We thank B. Canup for proofreading of this manuscript. We also thank Z. Liu, L. Yang, H. Mao, and A. Patel for helpful discussion on this project. Funding: This work was supported by NIH research grants (AA112713 and CA183376) to J.J.Y. and Georgia State University Brain & Behavior fellowship to S.T. Author contributions: Conceptualization: S.T. and J.J.Y.; formal analysis: S.T., H.Y., S.X., M.S., R.M., Y.H., P.Z.S., P.M., H.E.G., and J.J.Y.; investigation: S.T., H.Y., J.Q., W.H., F.P., K.H., Y.M., and O.Y.O.; methodology: S.T., H.Y., S.X., and J.Q.; writing (original draft): S.T.; writing (review and editing): S.T., D.L., V.M.M.-T, M.L.Y., and J.J.Y. Competing interests: F.P., S.X., and M.S. are inventors on a patent related to this work filed by Georgia State University Research Foundation Inc. (no. WO2016183223A2, published on 17 November 2016). The authors declare no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper maybe requested form the authors.

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Chemokine receptor 4 targeted protein MRI contrast agent for early detection of liver metastases - Science Advances

'This is the beginning of my new life'

I thought my cancer diagnosis was a death sentence, said Lynnette Williams-Briggs, 60, of Seaford, Delaware, who was diagnosed with advanced B-cell lymphoma in 2018.

Briggs cancer is now in complete remission thanks to successful chimeric antigen receptor CAR-T cell therapy she received in August atChristianaCaresHelen F. Graham Cancer Center & Research InstitutesBone Marrow and Stem Cell Transplant Program.

I can breathe again. This is the beginning of my new life, Williams-Briggs said following the treatment that restored her hope for a second chance at life.

She was the first patient to receive CAR-T cell therapy in Delaware. A second patient was treated in December 2019, and doctors are preparing several more patients for CAR-T cell transplants in coming weeks.

The U.S. Food and Drug Administration has approved CAR-T cell therapy to treat patients like Williams-Briggs with highly resistant, B-cell blood cancers, for whom other available options have failed.

CAR-T cell therapy is only available at select cancer centers with specialized expertise in cellular therapies that are recognized for quality by the Foundation for the Accreditation of Cellular Therapy.

The Graham Cancer Centers Bone Marrow and Stem Cell Transplant Program is the only one in Delaware that is certified to treat adult patients with advanced B-cell lymphomas and children and young adults (to age 25) with acute lymphoblastic leukemia, using an FDA-approved drug.

CAR-T cell therapy is highly personalized medicine that attempts to use the bodys natural defenses to fight against cancer. The transplant team extracts millions of T cells, from the patients bloodstream, using a specialized blood filtration process called leukapheresis. The collected T cells are flash-frozen and sent to a lab for reprogramming, and then later infused back into the patient using a process similar to a blood transfusion.

The therapy is considered a living drug with potential benefits that could last for years.

When we first met Ms. Williams-Briggs, her cancer had progressed rapidly despite a third round of chemotherapy, so we knew we had to move quickly, said Graham Cancer Center Hematologist Peter Abdelmessieh, D.O. He worked closely with the bone marrow/stem cell transplant team and Graham Cancer Center leadership over the course of just eight months to develop the CAR-T cell therapy program.

It was truly a team effort to bring CAR-T cell therapy to our community so quickly, Dr. Abdelmessieh said.

CAR-T cell therapy has been extremely effective for many patients like Williams-Briggs, whose PET scan at 90 days confirmed her remission.

The supercharged T cells Williams-Briggs received were genetically modified in the lab to sprout new surface tools that improve their ability to recognize, latch onto and destroy other cells (including cancer cells) that express a specific antigen called CD19. These reprogrammed cells continue to multiply in the body after treatment, remaining on guard to seek and destroy any new cancers that might develop.

With continued success in increasing numbers of patients, it is conceivable that in the not too distant future, CAR-T cell therapy could become the new standard of care, replacing chemotherapy and stem cell transplants for many cancers, Dr. Abdelmessieh said.

The extended recovery period for CAR-T cell therapy is generally two to three months. After the infusion, patients may spend up to three weeks in the hospital to monitor treatment response and any side effects.

During the first 30 days after leaving the hospital, patients are required to remain close to the treatment center for regular follow-up care.

The ability to offer potentially life-saving CAR-T cell therapy is one more reason our patients need not travel further than the Graham Cancer Center for state-of-the-science cancer treatment, said Nicholas J. Petrelli, M.D., Bank of America medical director of the Helen F. Graham Cancer Center & Research Institute.

The Bone Marrow and Stem Cell Transplant Program is an outstanding example of how well our clinical teams work together to drive innovation in patient care.

Although patients normally do not experience the side effects associated with chemotherapy, such as nausea, vomiting or hair loss, CAR-T cell therapy is not without risks. A common side effect, which Williams-Briggs also experienced, is cytokine release syndrome. This is an inflammatory condition that causes flu-like symptoms that may be mild or severe.

The transplant team responded quickly to manage her symptoms while she received expert care on the Bone Marrow Transplant and Oncology unit at Christiana Hospital.

From the moment I first met with my transplant team, I felt like I was part of one big loving family that extended beyond my own loved ones, Williams-Briggs said.

Dr. Abdelmessieh and my ChristianaCare family gave me hope to keep fighting when I really didnt think I would make it. I would have driven anywhere to get life-saving treatment, but I am thankful that I did not have to. I found my miracle closer to home.

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First CAR-T cell cancer therapy patient in Delaware - Dover Post

A new desert healthcare center is opening to enhance the wellbeing of residents in the Coachella Valley.

HALO Dx, Inc. will enable valley residents to have quick and easy access to advanced diagnostics. This, in turn, will empower patients and permit more effectiveness in their medical treatment.

Today HALO Dx held a ribbon-cutting ceremony with the Indian Wells Chamber of Commerce at the Indian Wells office of HALO Dx.

State-of-the-art equipment and university-level services available at HALO Dx include: Magnetic resonance imaging (MRI) scans Computerized tomography (CT) scans Positron emission tomography-computed tomography (PET/CT) scans Ultrasound scans Early diagnosis of dementia and Alzheimers Biopsies and aspiration Circulating Tumor Cells (CTC) Coronary CT angiography Facet injections Genomics Interventional radiology and pain management procedures Lung cancer screening Multiparametric MRI (mpMRI) for prostate cancer detection, diagnosis and treatment Nuclear medicine Virtual colonoscopy Whole-body MRI screening Clinical trials for conditions including prostate, lung cancer, stem cell and other studies Personalized healthcare using emerging genomic solutions

In the near future, HALO Dx will also offer a womens health imaging program, radiology A.I. solutions and expanded nuclear medicine and genomics services.

For more information about HALO Dx, call 760-275-5768, or visit HALODX.com.

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HALO Dx Center Offers Innovative Healthcare to the Coachella Valley - NBC Palm Springs

A year ago Janice Baker's future seemed bleak.

A second bout of chemotherapy in February 2019 had failed to erase her cancer.

She was so weak she could not walk.

Janice, from Romiley in Stockport, had been diagnosed with large B-cell lymphoma a type of cancer of the blood and lymph glands in April 2018 after feeling unwell for several months.

But now, she has the vim and energy to look after her grandchild after a remarkable response to a pioneering treatment.

CAR-T cell therapy helps to genetically reprogramme a person's immune system to help them fight cancer.

Janice was the first patient to undergo the therapy at Manchester Royal Infirmary (MRI).

Manchester University NHS Foundation Trust (MFT) was named as one of a very small number of centres in the UK to offer the revolutionary new treatment, widely regarded as the most exciting cancer treatment development in decades, in 2018.

Janice underwent two separate bouts of chemotherapy in August 2018 and February 2019, which didnt manage to successfully treat the cancer.

It was then she was offered CAR-T therapy.

Janice, who was diagnosed with the cancer in the same week her husband Chris was diagnosed with prostate cancer, said; Over the last 18 months my health was very up and down. At my worst I could barely get out of bed. I had to use a wheelchair, lost a lot of weight and hardly left the house, except for hospital appointments.

When I heard I was eligible for CAR-T cell treatment I was thrilled as I had read up about the amazing results and it felt like a real lifeline. My chemotherapy had limited success which was unfortunate but did mean that I was a bit stronger to face the CAR-T treatment.

The treatment itself was actually quite straightforward. The removal and replacement of cells is not that intrusive, and the wonderful staff were very attentive and kept a very close eye on me. There can be a few side effects but, fortunately, I had very few problems and the after effects were quite minimal.

Janice had the CAR-T treatment in May 2019 and was an inpatient for a couple of weeks at the MRI before returning home.

CAR-T (Chimeric Antigen Receptor T-cell) therapy is a personalised medicine used to treat patients with certain types of leukaemia and lymphoma.

It is a highly complex new type of immunotherapy which involves collecting and using the patients own immune cells to target their cancer in a process which is completed over a number of weeks.

Janice added; After two weeks I was feeling encouraged that everything was going well. I was still quite poorly and it has been a long slog but I have slowly been building up my strength.

I had a scan in August, which we hoped would show a significant decline in the cancer, but the results amazed both the doctors and ourselves, as it showed that the cancer cells seem to have gone completely.

Things are now getting back to normal. I have been on holiday, am back to driving my car and, best of all, I have the energy to look after my Grandson. I really feel like I have the opportunity to start planning for the future again.

I had another PET scan in November 2019, which was also clear, so I am feeling really optimistic now.

Dr Eleni Tholouli, Consultant Haematologist and Director of the Adult Stem Cell Transplant and CAR-T Therapy Unit at Manchester Royal Infirmary, and who has been involved in Janices care throughout, said: We are delighted for Janice and her family that she has responded so well to CAR-T therapy. This is a ground-breaking new treatment for adult cancer patients at the MRI which uses the patients own immune cells, allowing us to create a powerful medicine tailored to an individuals needs.

So to see Janice have these incredible results in such a short space of time is really encouraging. This reassures us that we have to continue all the hard work and make this therapy available to more patients.

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"The results amazed both the doctors and ourselves" - first patient to receive pioneering cancer treatment at MRI - Manchester Evening News

Every year, the equivalent of the total population of Tuscaloosa, Alabama slightly more than 100,000 people die worldwide as a result of snake bites. Provided a snake bite victim is able to get to the emergency room quick enough, antivenom can be used to counter the deadly effects of a bite. But antivenom isnt easy to manufacture. Its made by collecting venom from venomous snakes and injecting small quantities of it into a domestic animal such as a horse. The antibodies that form can then be collected from the horses blood and purified to make a finished antivenom. So far, so straightforward.

The problem is getting hold of enough venom to make it. Antivenom is currently manufactured by catching or breeding snakes, keeping them in captivity, and then regularly milking them to gather the venom they produce. Its a 19th century treatment thats made necessary by the fact that antivenom production has not developed as fast as other areas of biotechnology. With 600 species of venomous snake, its also a labour-intensive job which nonetheless struggles to create antidotes enough to meet the number of annual snake bites. Could genetic engineering be the answer?

A group of three researchers at Utrecht University in the Netherlands think so. And their idea for achieving it is kind of brilliant. Rather than creating lab-grown venomous snakes an idea that, frankly, would only sound good to one of the screenwriters of Sharknado theyve come up with an alternative solution: Simply grow the part of the snake that you need.

We were thinking about novel areas for [our] organoid technology, Hans Clevers, whose lab carried out the work, told Digital Trends. Snake venom glands were the most fascinating tissue to us. A main first hurdle was to obtain snake tissue. Luckily, a collaboration with snake experts Michael Richardson and Freek Vonk, as well as the Dutch reptile zoo Serpo and local breeders solved this issue. After some months of optimizing the protocols, we were successful in growing miniature venom glands. Since then, we have been optimizing the protocol to produce venom and have characterized the cells which make the toxins.

Aspidelaps lubricus hatching (individuals not used in study) Jeremie Tai-A-Pin

An organoid, for those unfamiliar with it, is a miniaturized and simplified version of an organ, complete with realistic micro-anatomy. Theyre made using stem cells, which let them self-organize in a three-dimensional culture to transform into the organ theyre supposed to replicate. The emerging organ is a clump of cells around 1 millimeter across. Organoids have been created by various labs around the world, approximating organs that range from kidneys to miniature, non-conscious brains. In this case, the venomous organoids resemble a tiny pea-sized balloon filled with liquid. One that would be particularly inhumane to fire across the classroom like a spitball wad.

Our group has been successful in the past 10 years in growing organoids from a variety of human tissues, Joep Beumer, another researcher on the project, explained. To generate these, we harvest stem cells from adult tissue and embed them into a gel in a petri dish. With the right growth factor mix, the stem cells will divide and give rise to mini-organs containing the different cell types of one tissue.

The tissue samples for the venom organoids were taken from gland tissue from snake embryos inside eggs or, in one instance, from a pet snake which had been put down as a result of illness. To grow the gland organoids, the team had to make a few changes to their normal approach. Snakes are cold-blooded. Mammalian organoid protocols are normally grown at a temperature of 37 degrees Celsius (99 degrees Fahrenheit). Unfortunately, this didnt work for the snake organoids. At this temperature, the organoids suffered heat shock response and died. As a result, they had to lower the temperature to 32 degrees Celsius. Its a demonstration of how, even at this scale, the concepts and signaling pathways of adult stem cells are conserved in organoids.

Every tissue has its own characteristics which we aim to model with organoids, said Yorick Post, the third researcher on the project. For the snake venom gland this was a very obvious case: would they make venom? We knew that the potential of this technology would hinge greatly on the ability to produce the different toxins which constitute snake venom. So we were very excited when we found toxins first on RNA, and later on [the] protein level.

This work is extremely promising. The researchers think it could potentially go beyond just cutting out the snake-farming part of the antivenom process as well. They believe it might be possible to grow the immune cells that are usually produced by animals inside a dish. Alongside antivenom, the approach could also be useful for helping develop drug compounds based on components found in snake venom. For instance, theres a certain type of blood pressure medicine thats created from a toxin produced by the venomous Brazilian pit viper.

And as to, no pun intended, the scaling up part of the project? This is one of the main advantages of organoid technology, Clevers said. Once established, we can expand the tissue [in a] pretty much unlimited [manner]. This can help to preserve viable cells of many snake species, as they can be frozen and thawed easily. [It can also help us] generate large numbers of venom producing cells. Further improvements in venom production and harvesting will be needed to make this approach cost efficient. We are actively working towards these aims.

A paper describing the work was recently published in the journal Cell.

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Lab-grown snake venom glands are here. Dont worry; theyre for a good cause - Digital Trends

Technological advancements have paved the way for many important breakthroughs in biomedical engineering. New methods are being developed, as are our understanding, diagnosing and treating of medical conditions.

Unsurprisingly, the job outlook for biomedical engineers looks promising. The US Bureau of Labor Statistics notes that employment of biomedical engineers is projected to grow four percent from 2018 to 2028, about as fast as the average for all occupations. It adds that the increasing number of technologies and applications to medical equipment and devices, along with the medical needs of a growing and ageing population, will further require the services of biomedical engineers.

If youre trained in biomedical engineering or are a graduate in a related field looking to enhance your qualifications or progress into a leadership role, you may want to consider enroling in doctoral studies in biomedical engineering.

A good place to start is Michigan State University (MSU), which has carved itself a strong reputation in the field.

Its Biomedical Engineering Department (BME) offers a competitive research-oriented doctoral programme with flexible and personalised curricula.

The department is housed in a state-of-the-art research facility and engages with faculty across several disciplines, departments and colleges to explore the intersection of medicine, human biology and engineering.

The BME department is housed within a new research facility, the Institute for Quantitative Health Science and Engineering (IQ). IQ consists of seven research divisions, i.e. biomedical devices, biomedical imaging, chemical biology, developmental and stem cell biology, neuroengineering, synthetic biology and systems biology.

The interdisciplinary research centre is devoted to basic and applied research at the interface of life sciences, engineering, information science and other physical and mathematical sciences.

Students have access to the stellar facilities and equipment at IQ, which foster extensive collaboration between researchers from different areas to solve some of the worlds most challenging biomedical problems.

This systems approach to biomedical research look set to lead to discoveries that are the first of their kind. IQ is connected to both the Clinical Center and Life Sciences buildings, establishing a biomedical research hub at MSU that holds the potential to transform medicine.

The BME department also boasts a range of expertise, including advanced imaging methods and nanotechnology in biomedical research.

Training PhD students in the biodesign process is a priority here whereby students identify significant needs for new biomedical technologies before developing commercialisable technologies that meet those needs.

MSU also provides a host of services to help students healthcare solutions make it to market.

The MSU Innovation Center houses MSU Technologies, Spartan Innovations and MSU Business CONNECT in support of entrepreneurship, facilitating technology transfer, and providing the educational and financial support to turn doctorate students research technologies into successful businesses.

Another major focus of the BME department is biomedical imaging, including the development of new nanoparticle-based combined imaging and therapeutic technologies. The IQ building has one of the few PET MRI systems in the world.

What differentiates MSU from other institutions is their new, two-semester course sequence on the development and translation of new biomedical technologies to meet clinical needs.

Named BioDesign IQ 1 and 2, these courses train BME PhD students and professional students from the colleges of medicine, law, and business to work together effectively in innovation teams. They shadow doctors, identify unmet medical needs that have significant market potential, prototype new technologies to meet those needs, and then develop intellectual property and a business plan to advance these new technologies towards commercialisation.

Apart from its stellar facilities, the university is also home to faculty with strong expertise.

For instance, inaugural IQ director and BME chairperson Christopher H Contag is a pioneer in molecular imaging and is developing imaging approaches aimed at revealing molecular processes in living subjects, including humans and the earliest markers of cancer. Through advances in detection, professionals in the field can greatly improve early detection of diseases and restoration of health. Contag was previously at Stanford University as a professor in the departments of Pediatrics, Radiology, Bioengineering, and Microbiology and Immunology.

Meanwhile, Dr Mark Worden, BME Associate Chair, has developed several interdisciplinary programmes that integrate research and education. His research on nanostructured biointerfaces and multiphase biocatalysis has resulted in over 10 patents issued or pending on technologies including microbiosensors, bioelectronics and multiphase bioreactors.

Source: Shutterstock

Other faculty members doing trailblazing work in the field include Dr Dana Spence, who is investigating and dening new roles for red blood cells in autoimmune diseases such as Type 1 diabetes and multiple sclerosis; Dr Aitor Aguirre, whose research focuses on investigating regeneration and tissue re-modelling in health and disease; and Dr Ripla Arora, who is working on understanding how hormones influence the uterine luminal and glandular epithelium to modulate receptivity and implantation, to name a few.

In addition to insightful guidance from a faculty of this calibre, PhD students also enjoy 100 percent funding, including stipend, tuition and healthcare. As a graduate student in biomedical engineering, they will have the valuable opportunity to work alongside graduate students from different departments across campus.

Without a doubt, a PhD in biomedical engineering from MSU will prove to be fulfilling endeavour, professionally and personally.

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4 leading North American universities for biomedical engineering

Humanitas MEDTEC School: Where science and biomedical engineering meet

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Engineer the future of human health with a PhD in biomedical engineering - Study International News

Dr. Dori Borjesson

PULLMAN, Wash. Dr. Dori Borjesson, chair of the Department of Pathology, Microbiology and Immunology at the University of California Davis School of Veterinary Medicine, has been selected as the new dean of the WSU College of Veterinary Medicine.

Borjesson was chosen following a nationwide search to replace Dr. Bryan Slinker, who had announced plans to retire before being tapped to serve as interim provost. She will assume her new responsibilities leading WSUs cutting-edge veterinary, biosciences and global health departments on July 20.

The strength of Washington State Universitys research and its potential to impact communities locally and across the globe impressed me during the interview process, as did its dynamic clinical programs and the Washington-Idaho-Montana-Utah Regional Program in Veterinary Medicine, Borjesson said.

Im looking forward to building on Dr. Slinkers tremendous tenure of leadership, she continued. The enthusiasm for WSU among the community is impressive, and I look forward to building on that momentum.

In addition to her role as a department chair and full professor at UC Davis, Borjesson works as a clinical pathologist and is actively engaged in clinical service and laboratory test development. She served as the inaugural director of the Veterinary Institute for Regenerative Cures from 2015 to 2019 and continues to direct the Clinical Regenerative Medicine Laboratory.

Dr. Borjesson brings an important combination of strengths and experience to make her the right leader for the college, Slinker said. Shes a long-serving, highly regarded, and very effective academic leader, and an excellent clinician/scientist, at an aspirational peer institution. This background, combined with her intellectual rigor, openness, and compassion make her a great fit to lead the college in its next phase of growth and development as one of the nations top veterinary colleges.

Borjesson said shes thrilled to meet with WSU students, staff and faculty, as well as meeting with college and university stakeholders in the near future.

Being from the Pacific Northwest, this feels like a homecoming, said Borjesson, who was raised in Portland, Ore. Increasing engagement and outreach across the state is a top priority for me upon taking up this new role. In addition to engagement and strategic planning, Im also eager to face some of the critical issues facing members of the veterinary profession, including student debt and enhancing the well-being of our faculty, students and staff.

Among her more notable research contributions is using large animal models of disease to study cell therapy for inflammatory diseases.

Borjesson holds two patents in the area of mesenchymal stem cells and immunomodulation and has contributed to more than 100 peer-reviewed publications, and in 2014 received the Zoetis Research Excellence Award. Alongside her own work, she has mentored more than three dozen veterinary residents and graduate students.

She and her colleague Dr. Aijun Wangs work with stem cells was highlighted in an extensive piece in the Los Angeles Times in 2018 about UC Davis Veterinary Medical Teaching Hospital.

Borjesson received her undergraduate education from the Colorado College in 1988, her Master in Preventive Veterinary Medicine and Doctor of Veterinary Medicine degrees from UC Davis in 1995. She completed a residency at UC Davis in clinical pathology in 1999, followed by her PhD in comparative pathology at the Center for Comparative Medicine at UC Davis in 2002.

After completing her PhD, Borjesson accepted an assistant professorship at the University of Minnesota, where she worked for four years before returning to UC Davis as an associate professor in 2006. She became a full professor in 2012. She has led the Integrative Pathobiology Graduate Group at UC Davis and is actively engaged in veterinary and graduate student curriculum development, teaching and mentoring.

Established in 1899, the WSU College of Veterinary Medicine is proud of its distinguished past as one of the oldest veterinary colleges in the United States. It is equally proud of its contemporary leadership nationally in offering programs for student wellness, its Teaching Academy, which leads its commitment to advancing the state of the art in both health professions and STEM education, and its research and graduate education programs. The breadth of research to discover foundational knowledge and to conduct research targeted to improve animal and human health both domestically and around the world places it in the top 10% of veterinary colleges in receipt of competitive federal research funding.

Phil Weiler, vicepresident for marketing and communications, 5093351221, phil.weiler@wsu.edu

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Dr. Dori Borjesson named dean of the WSU College of Veterinary Medicine - WSU News

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While the coronavirus is currently not a public health emergency, as per the World Health Organization, its not impossible that a novel disease could get out of hand in a hurry. Im writing this article because, amidst all the coverage of the Wuhan virus which has quarantined 35 million and counting, I watched Contagion, thus spooking me out tenfold.

Contagion, a 2011 movie starring Matt Damon about an unknown virus that starts in China thanks to bat droppings and creeps its infected hands across the globe, paints a horrifying picture of what can happen when scientists arent able to keep up with a disease. (An eerily similar situation to whats going on now, though fairly easy to predict granted Chinas history with diseases.)

And while we are still nowhere close to pandemic levels, its still important to know what to do in a situation where the virus has landed in your country and is potentially making its way through your neighbourhood.

Before delving in, though, I want to say: do not panic, this is a hypothetical article about a very specific scenarioone that humanity has gotten pretty good at curbing. With that said: here are 10 steps you should take to minimize your chances of contracting a novel virus if things were to get that bad.

*Note, I am NOT a medical professional. These suggestions are a collection of several health websites throughout the web.

In some densely populated Asian countries, its not uncommon to see citizens wearing white medical masks on their faces, and in the videos circulating online of Wuhan, youll be quick to see just about everyone wearing one. This is because protective masks, while not fool-proof, can decrease your chances of breathing in air-borne projectiles through coughs or sneezesif applied properly.

Professor of molecular Jonathan Bell at the University of Nottingham has said: In onewell-controlled study in a hospital setting, the face mask was as good at preventing influenza infection as a purpose-made respirator. So strap up!

One of the best things anyone can do to stop the spread of diseases is thoroughly washing your hands with soap and warm water. In times of real strife, its advised by the CDC that you wash your hands.:

While that may seem obsessive, take this as an example: In developing countries, childhood mortality rates related to respiratory and diarrheal diseases can be reduced by introducing simple behavioural changes, such as handwashing with soap. This simple action can reduce the rate of mortality from these diseases by almost 50 percent.

All of this is hand-in-hand with not touching your face. The average person touches their face 23 times an hour. Avoid scratching or rubbing your face or nose with your hands, unless recently washed.

Public transportation is a notorious playground for bacteria and diseases to make their way.

The combination of hoards of people, all tightly packed in tubes and all touching the same handles and doors is not ideal when avoiding an illness. But, there are a few steps you can take to make the ride a bit easier on you:

Gloves, though they do need to be changed fairly frequently, are a highly effective way to avoid bodily fluids. Saliva, the main culprit, can be spread easily via coughs and sneezes into hands, and then on to public transport. This is why the sleeve sneeze, or the vampire sneeze, is another great method to avoid getting others ill.

Other, more obvious bodily fluids to avoid include blood, vomit, urine, and feces, which all pose a higher risk of cross-contamination.

Pro tip: Avoid wearing gloves while preparing food. While this may seem like a good idea, it may actually make the odds of cross-contamination more likely. This is why many professional kitchens will opt for frequent washing rather than gloves.

The little things go a long way, whether its precautions or bacteria. There are several little things that can make a big difference, including alcohol wipes for cell phone screens. Cell phones are an often overlooked way of spreading bacteria. Avoid voice calls on your cell phone unless youve got a way to disinfect your screen first.

Other things you can do if youre particularly vigilant are avoiding the handling of cash, and not allowing others to handle your debit card.

These suggested steps are still a bit further ahead in the future than the state we are currently in with coronavirus, and lets hope we dont ever need them. The last few notable public health crises, (Ebola, Zika, H1N1) did do significant damage in given regions, but were all eventually contained and are all no longer considered public health risks and are now at what is considered normal levels.

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Five tips to stay healthy during a coronavirus outbreak - The Post Millennial

Getting a puppy is an exciting time for a pet parent. Usually, people are excited to live in the moment with a new member of the family.

Just like with a human baby, however, you need to take the future into consideration. For pet parents, that means checkups, pet insurance, and getting the little one spayed or neutered. A new service from Gallant, an animal biotech company on a mission to help pets live healthier and happier lives, offers an additional consideration stem cell banking for your pet.

Gallants stem cell banking service launched just a few weeks ago, but its already creating some major buzz.

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The company has acquired the veterinary division of Cook Regentec. The acquisition includes its intellectual property, existing stem cell banking operations, and pipeline of cell therapy products derived from reproductive tissue. Its patent-pending process allows stem cells to be collected at the time of a standard spay or neuter procedure, which is great news for pet parents who dont want to put their fur babies through any further invasive procedures.

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Pet Owners Can Collect And Bank Their Pet's Stem Cells ...