As well as the challenge of creating new vaccines, there were vexing problems with existing ones. One of two existing rabies vaccines, made in dried animal brains, could produce fatal allergic reactions; the other, made in duck embryos, was not as effective as the animal brain–produced version. And a silent monkey virus had been discovered in monkey kidney cells used to make the Salk polio vaccine. As he prepared his paper in the autumn of 1960, Hayflick, unlike the public, was keenly aware of the monkey-virus problem.
It wasn’t lost on Hayflick that his human fetal cells might provide clean, safe alternative microfactories in which to produce viral vaccines, if—and it was a big “if”—they could be infected with disease-causing viruses. There was precisely one way to find out if that was the case. Hayflick began infecting bottles of the WI cells with different viruses. They turned out to have a huge range of virus susceptibility: thirty-one viruses invaded and damaged the cells. These viruses included measles, rabies, herpes simplex, adenovirus, influenza, and polio. The cells even succumbed to invasion by varicella, the virus that causes both chicken pox and shingles but is extremely choosy about which cells it will grow in.18
The implications were exciting, at least in Hayflick’s eyes. Public and scientific interest in antiviral vaccine making was surging. At this juncture it could be a big advance to introduce a new, safe, plentiful supply of cells for vaccine making. But Hayflick also knew that it was going to be an uphill battle to convince vaccine regulators that his cells were safe. After all, the hot area in cancer research was the purported potential of as-yet-unidentified hidden viruses to cause cancer. Vaccine-approving agencies were going to want assurances that no such unidentified viruses lurked in his human fetal cells. Nothing would put them off the cells more quickly than the possibility of such a virus getting into a vaccine and later causing cancer in vaccinated people.
Hayflick examined his two dozen fetal cell lines under the microscope repeatedly. He found no telltale signs of viral infection. He took the fluid bathing the cells and injected it into cultures of other kinds of cells, and into animals. Neither the cells nor the animals showed any signs of infection.19
Then he turned again to Moorhead to scrutinize the cells’ chromosomes. Virtually all cancers had abnormal-looking chromosomes and abnormal numbers of chromosomes. If any of the WI cell lines showed such deviations, that too would mean a no-go from regulators. Their thinking would run like this: If the fetal cells harbored abnormal chromosomes, they were either cancerous or would soon become so. If this was the case, then there was every chance that a hidden virus in the cells had caused the malignant changes. And if a hidden, cancer-causing virus was in the cells and they were used to make a vaccine—well, there was an epidemic of cancer just waiting to happen.
Hayflick waited while Moorhead stared at sample after sample of the fetal cells, counting chromosomes painstakingly, hour after hour, week after week. He looked at young cells, which had divided just nine times. And he was careful to look at old ones too: the oldest cells he examined had been through forty replications. Since chromosomes were particularly vulnerable to developing cancer-associated abnormalities during cell division, the older cells, having divided more times, were most at risk of showing anomalies.
When Moorhead finally looked up at the end of weeks of work, in the autumn of 1960, he had good news for Hayflick: these cells, young and old alike, were normal cells, not aberrant ones. They were diploid cells, with twenty-three pairs of chromosomes, for a reassuring, normal total of forty-six. And so Hayflick dubbed the fetal cells “human diploid cell strains.” The term “cell strain” was a very deliberate substitution for “cell line” on Hayflick’s part. In Hayflick’s nomenclature a “line” is a group of cells that will go on dividing endlessly, whereas a “strain” denotes a group of cells that is mortal—that will reach the Hayflick limit and then expire.
Hayflick was now armed with reassuring chromosome data, which Moorhead documented with striking photos showing the chromosomes of several of the WI lines neatly laid out in twenty-three numbered pairs. But Hayflick wasn’t finished amassing evidence for the safety of the cells. He enlisted a scientist friend of his, Anthony Girardi, who worked at Merck in nearby West Point, to conduct another key experiment, this time on hamsters—an additional study aimed at persuading regulators that these cells were not cancers-in-waiting.
Hamsters’ big cheek pouches don’t have the normal immunological defenses that attack foreign invaders, including cancer cells. That makes them useful to biologists, who, as Hayflick worked, had already shown that cancer cells formed ever-enlarging tumors when they were injected into hamster cheek pouches. Hayflick’s friend Girardi injected the animals’ cheek pouches with vigorously growing WI-25 cells. If the cells had any propensity to turn into cancers, several weeks growing in hamsters’ cheeks would give them plenty of opportunity to do so. For comparison, Girardi injected five additional, control hamsters with aggressively cancerous HeLa cells.20
The HeLa-injected animals sprouted tumors in their cheek pouches, and those tumors were still growing after three weeks. None of the WI-25–injected animals developed cancer.21 These findings were reassuring, but for Hayflick they still weren’t enough to counter the fears he anticipated among vaccine regulators. Hamsters were not human beings. The cells, he determined, had to be put into people.
Hayflick believed that the risks of such an experiment would be minimal. He was aware that a few years earlier, a high-profile cancer researcher named Chester Southam had taken microscopically normal-looking cells—fibroblasts, the same cell type as Hayflick’s fetal cells—from a human embryo and injected them under the skin of three dying cancer patients whom he described as “volunteers.” Southam, who was chief of virology at the prestigious Sloan-Kettering Institute for Cancer Research in New York City, reported that the embryonic cells had not grown in the dying patients, unlike the cancer cells that he had simultaneously injected in the same patients.22
Southam’s name has come to be notorious among medical ethicists, and his studies, which also included injecting aggressive cancer cells into healthy prisoners at the Ohio State Penitentiary, would ultimately lead to a public outcry in 1963. That year three doctors at the Jewish Chronic Disease Hospital in Brooklyn, New York, would resign in protest rather than inject unsuspecting patients with cancer cells for Southam.23 Their action launched a lawsuit, a state attorney general’s investigation, and a media storm that both accelerated and reflected changing public perceptions. Southam ended up being put on probation by New York’s medical licensing authority for one year. Many in the medical establishment were not convinced that he had done anything wrong, and soon after his probation ended he was elected president of the American Association for Cancer Research.24 But by the mid-1960s, ordinary people were becoming less willing to give scientists carte blanche to tinker with human beings on a “Trust me, I know what’s best for you” basis.
That was not the case in 1957, which is the year that the Southam study injecting the normal-looking embryonic cells, along with cancer cells, in dying cancer patients was published in Science, the leading American science journal. The notion that dying patients, who had nothing to gain by participating and who were enlisted in a study by their doctors, who had everything to gain by their participation, could be called “volunteers” was accepted then. So was the practice of putting such patients at risk. Southam implied as much when he wrote in Science that they “had advanced incurable cancer and a very short life expectancy.”25 And so, as they planned for the injection of their own human fetal cells into dying cancer patients, Hayflick and Moorhead felt not only that they were doing nothing wrong, but also that they were following in the footsteps of an eminent man of science. Indeed, their paper would state that Southam’s experiment had laid the groundwork for their own.26
It made sense for them to turn to Robert Ravdin, the son of the powerful HUP surgeon in chief I. S. Ravdin, for help inject
ing the diploid cells into dying cancer patients; Ravdin specialized in cancer surgery. A comment that he made in this era suggests that he likely had no compunction about the injections. In 1964, when Chester Southam was on the public hot seat, Robert Ravdin, defending Southam, would tell a reporter that if every subject in a human trial had to be fully informed, everyone would need a PhD.27
Being a hard-charging surgeon—a prince of the hospital, if not the king—the junior Ravdin did what surgeons do. He delegated the task downward, to a second-year surgical resident named William Elkins. The twenty-eight-year-old Elkins had an impeccable pedigree: he had attended Princeton as an undergraduate, then gone on to Harvard Medical School. But as a surgical resident at HUP, he was becoming convinced that he was not cut out for the punishing, testosterone-driven world of the operating room. What he wanted to do was science. The next year he would move to the Wistar and begin a long research career in transplantation immunology.28 As Thanksgiving 1960 approached, he was still slogging it out on the surgical wards when his boss, the junior Ravdin, asked Elkins to do a menial chore: inject some fetal cells belonging to Wistar scientists into a few dying patients.
It’s not clear if, how, or when these patients were informed about the experiment or what they understood of the process and its purposes. Both Hayflick and Elkins, recalling these events more than fifty years later, conjectured that Ravdin approached the patients and explained the experiment. However, it would emerge in the mid-1960s that in dozens of studies in this era patients were not informed that they were experimental subjects. It seems at least possible that that was the case here.
Hayflick chose WI-1 cells for the injections. These were not young cells, and he wanted it that way. If the WI cells were going to morph into cancer cells, these older cells were the likeliest to do so. Conversely, if these aged cells weren’t cancerous, his case that they were normal would be all the stronger for the fact that he had used older cells.
Hayflick gave Elkins two lots of WI-1 cells. He had grown both of them, then frozen them for months, and then thawed them and grown them some more. One group had divided thirty-seven times. The other was still older, having replicated forty-five times.29 If any one of the cells was cancerous, he would expect it to form a tumor at the injection site. A biopsy of the tumor would reveal abundantly growing cells with the microscopic hallmarks of cancer.
One day late in 1960, Elkins put a small syringe fitted with a fine needle on a tray alongside a test tube containing a sterile solution of salts and water. In each ounce of that saline solution there were some 177 million living WI-1 cells. Then he headed to the surgical wards.
Visiting a dying cancer patient, Elkins turned one of the patient’s forearms over, revealing its softer underside. He swabbed the skin with disinfectant and then turned to his tray. He sucked half a milliliter—less than two hundredths of an ounce but containing about three million WI-1 cells—into the syringe, eased the needle under the patient’s skin, and pushed the plunger. Then, using a marker, he drew a circle around the injection site. He would be back to check the result, he told the patient.30
This faceless patient and five others are identified only by their initials in Hayflick and Moorhead’s landmark paper. In it there is a table describing the injections and their results.31
The first patient had cancer that had spread throughout his or her abdomen. The doctors were at the point of simply treating symptoms, not trying to stop the disease. After the patient was injected, a nodule, or bump, developed at the injection site but disappeared on the sixth day. A biopsy of the nodule, to look at its cells under the microscope, was not done.
Three of the patients had breast cancer that had spread. All three were on drugs that suppressed their immune systems, making them less able to fight off WI-1 if it was in fact cancerous. One of them developed a slight fibrous hardening at the injection site on the seventh day; she died on the eighth day. A biopsy of her injection site was negative for WI-1 cells or anything else abnormal. A second developed a nodule on the sixth day that disappeared by the tenth, four days before she died. A biopsy wasn’t done. The third breast cancer patient didn’t develop a nodule. Nonetheless, her injection site was biopsied on the seventh day, with negative results.
The other two patients had lung cancer and colon cancer respectively. Their diseases had spread through their bodies and they were on chemotherapy. One developed a small nodule on the sixth day. A biopsy showed no WI-1 cells, normal or cancerous. As for the other patient, the paper simply says that nothing had happened at the injection site after nine days, and that no biopsy was taken.
The sum of the results was reassuring: of the nodules developed by four of the patients, three of them melted away, and a biopsy of the fourth showed no cells of any kind. And no nodules had appeared at the injection sites of the other two patients.
By the summer of 1962, another eleven dying cancer patients would be injected with Hayflick’s cells, bringing the total to seventeen. A report by Hayflick and others to the World Health Organization in July of that year states that the cells had been “implanted in 17 patients dying of cancer.” Again the nodules that developed in some patients disappeared within ten days, and biopsies didn’t reveal any living cells.32 The results from these seventeen anonymous patients would be a key basis on which Hayflick would testify innumerable times over decades that his human diploid cells were normal and didn’t cause cancer.
For Hayflick the injections by Elkins were the final experiment in what was now eighteen months of painstaking trial-and-error work—work that had tested not only his scientific thinking and resourcefulness but his, and Moorhead’s, capacity for repetitive, monotonous tasks and meticulous observation. He had established what were, to his knowledge, the first cell lines that had been proven to be, and to remain, certifiably normal when grown in the lab. They could be grown for months and months and yet they still reliably retained the diploid number of forty-six chromosomes. And those chromosomes, scrutinized by Moorhead under the microscope, looked just as they ought to: his diploid cells did not harbor the broken, disjointed, frayed, and otherwise strangely constructed chromosomes that signified cancer. Nor did they appear to cause cancer in hamsters or human beings.
And just as reliably as they harbored forty-six chromosomes, the diploid cells aged and finally died in culture; they could replicate only through fifty or so divisions. They were as mortal as all flesh. The two sets of observations—the cells’ normalcy and their inevitable deaths—he now knew, went hand in hand.
His findings could be summarized like this:
Normal (diploid) chromosome number and appearance = finite growth in the lab
A few years later he would take this thinking further:
Normal (diploid) chromosome number and appearance = finite growth in the lab ←→ corresponds to normal cells in human beings
Abnormal (heteroploid) chromosome number and appearance = indefinite growth in the lab ←→ corresponds to cancer in human beings33
Normal cells could escape from aging only by acquiring the properties of cancer cells—whether in the lab or in a living human being, he would write.*34
Hayflick had also created, he believed, a promising tool to deploy against infectious diseases. For he had demonstrated that his diploid cells could be infected with dozens of disease-causing viruses, making them near-perfect factories for making viral vaccines. They appeared to be clean—free of hidden, lurking, unknown viruses that would scare off regulators—and they could be produced and then frozen, thawed, and expanded into near-infinite quantities of cells for just such a purpose.
For one junior scientist and his chromosome-expert colleague, it seemed that there was a tremendous amount to be proud of in this paper, which, as Hayflick wrote it up, stretched to thirty-five pages. He knew precisely where he wanted to place it.
In late 1960 the Journal of Experimental Medicine was an enviable place for a you
ng, ambitious scientist to be published. Already sixty-five years old, which was a considerable age in the young world of U.S. research, the journal had been founded by William Welch, a giant of American medical research. It had published many of the great biologists. It had also published Carrel’s 1912 paper establishing, via the beating chicken heart, that cells could live indefinitely in a culture bottle if they were just treated properly. The journal was put out by what was arguably the pinnacle U.S. research institution: the Rockefeller Institute in New York City. There, none other than Peyton Rous presided over it as editor.
Rous was the biologist who, fifty years earlier, had discovered that he could cause a sarcoma, a malignant connective-tissue tumor, in chickens by infecting them with finely filtered fluid from chickens that already had such tumors.35 He had posited that an “agent” separate from the tumor cells caused the sarcoma; since then this “agent” had been named the Rous sarcoma virus. With the resurgence in the 1950s of the idea that viruses might cause cancer, Rous’s fame and eminence had grown. In 1966 he would be recognized with a Nobel Prize, fifty-five years after publishing his groundbreaking paper.
Before Hayflick put the bulky manuscript and its glossy accompanying photos in the mail, he did two things. According to Moorhead, he suggested to Moorhead that the two men flip a coin to determine whose name should go first on the manuscript. Moorhead remembers declining the offer, saying that Hayflick had done the lion’s share of the work and deserved to be in the prestigious place of first author.36
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