12.2: Cancer Cells in Culture - Biology

12.2: Cancer Cells in Culture - Biology

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The photographs (courtesy of G. Steven Martin) show mouse fibroblasts (connective tissue cells) growing in culture. The cells in the top photo show contact inhibition. Those below do not. The cells below are said to be transformed. These cells (called 3T3 cells) were not derived from a mouse cancer but were produced by laboratory treatment of normal cells. Radiation, certain chemicals, and certain viruses are capable of transforming cells. Although transformed cells are not derived from cancers, they can often develop into malignant tumors when injected into an appropriate test animal (like a nude mouse).

Normal cells are exceedingly fussy about the nutrients that must be supplied to them in their tissue culture medium.

Cancer cells (and transformed cells) can usually grow on much simpler culture medium.

Normal cells ordinarily have the normal set of chromosomes of the species; that is, have a normal karyotype.

Cancer cells almost always have an abnormal karyotype with

  • abnormal numbers of chromosomes (polyploid or aneuploid)
  • chromosomes with abnormal structure:
    • translocations
    • deletions
    • duplications
    • inversions

12.2: Cancer Cells in Culture - Biology

Ascorbate oxidizes in cell culture medium to generate a flux of H2O2.

The rate constants for removal of extracellular H2O2 are on average 2-fold higher in normal cells than in cancer cells.

The ED50 of high-dose ascorbate correlated with the ability of tumor cells to remove extracellular H2O2.

The response to pharmacological ascorbate in murine-models of pancreatic cancer paralleled the in vitro results.

Cancer Stem Cells

Farhadul Islam , . Alfred King-Yin Lam , in Oncogenomics , 2019


Cancer stem cells (CSCs) or cancer initiating cells are a subpopulation of cells that has the driving force of carcinogenesis. They exhibit distinctive self-renewal, proliferation, and differentiation capabilities that are believed to play a critical role in cancer initiation, maintenance, progression, drug resistance, and cancer recurrence or metastasis. Factors such as increased activation of drug-efflux pumps, enhanced capacity of DNA damage repair, dysregulation of growth and developmental signaling pathways, alterations of cellular metabolism, environmental niche, and impaired apoptotic response are attributed to CSCs in their resistance to the adjuvant chemoradiotherapy to cancer. The development of strategies targeting CSCs via drug transporters, specific surface markers, inhibiting signaling pathways or their components, and destroying their tumor microenvironment have multifocal effects that may improve the clinical outcome of patients with cancer.

Technologies for CTC isolation and identification

Circulating tumor cells (CTCs) with morphologic features similar to the primary solid tumor were initially discovered by Thomas Ashworth [19] through an autopsy of a cancer patient 150 years ago. A number of scientists have demonstrated that CTCs can be used as a predictor of clinical prognosis and treatment efficacy evaluation [20,21,22,23]. At first, scientists used the CellSearch system, which was the only device for CTC analysis approved by the United States Food and Drug Administration (FDA), to enrich and enumerate CTCs from peripheral blood. Finally, researchers discovered that the enumeration of CTCs is insufficient because variable phenotypes of CTCs in circulation have different potentials in tumor progress. Detailed developments [19, 24,25,26,27,28,29,30] in the history of CTCs are shown in Fig. 2.

Milestones of CTCs development history

Abnormal proliferation and metabolism of tumor cells, disorder and changes in the composition of cells, unnatural gene expression and modification, and synthesis and accumulation of polar particulate lead to changes in the physical and biological properties of CTCs. Scientists have developed technologies for enrichment, isolation, and identification of CTCs according to these physical and biological changes. The methods of technologies for CTC isolation from the review by Rubis [31], are referenced, but only the latest technologies are listed in Table 1. Methods to isolate CTCs developed rapidly with the emergence of the microfluidic chip system and nanotechnologies. Using engineered mouse models in cancer research, Hamza et al. [32] have solved the problem of having small total blood volume and rare CTCs using an optofluidic-based approach, eliminating confounding biases induced by inter-mouse heterogeneity. Antfolk et al. [33] have isolated breast cancer cells (MCF7) from peripheral blood with an efficiency of 91.8 ± 1.0% based on an integrated acoustophoresis-based rare-cell enrichment system combined with integrated concentration. Abdulla et al. [34] have introduced a cascaded microfluidic device that can separate 80.75% of human lung cancer cells (A549) and 73.75% of human breast cancer cells (MCF-7) from the human whole-blood system based on their physical properties within 20 min with a cell viability of 95% and 98%, respectively. Neves et al. [35] have constructed a glycan affinity-based microfluidic device for selective isolation of membrane protein O-glycan sialyl-Tn antigen (STn +), which are more sensitive than size-based microchips for CTC detection and are clinically relevant with metastasis in bladder and colorectal tumors. To conclude, technologies for enrichment, isolation, and identification of CTCs according to physical and biological changes both have limitations, such as low purity, low cell viability, and low intermediate throughput. It is urgent to integrate the best of these technologies to generate a new approach that yield high throughput and high purity. With the emergence of numerous technologies and platforms for isolating and further analyzing CTCs, physicians have realized the importance of CTCs as liquid biopsy and therapeutic target.

Malignancy-Related Anemia and rEpo Treatment

Anemia is an independent prognostic factor for survival in patients with cancer (56). The European Cancer Anaemia Study recently reported that 72% of patients with hematologic malignancies and 66% of solid tumor patients were anemic at some point during the course of their disease although the exact prevalence of anemia varies according to the type of neoplasm (57). The pathophysiology of malignancy-related anemia can be multifactorial. In addition to anemia of chronic disease, nutritional deficiencies, bleeding, hemolysis, bone marrow involvement with malignant cells, and chemoradiotherapy can all contribute to anemia.

There is evidence that anemia may be associated with a diminished response to radiotherapy (58), chemotherapy (59), and surgery (60). Although a direct relationship between acute anemia and intratumoral hypoxia has been reported (61), the effect of chronic anemia is more difficult to interpret (62). Brizel et al. (63) found only a weak correlation between anemia and poor tumor oxygenation in head and neck cancer patients and many nonanemic patients had hypoxic tumors. Tumor hypoxia is common among a wide variety of malignancies and is a factor associated with treatment resistance, aggressive clinical phenotype, and poor prognosis (64, 65).

Beyond correcting anemia in preclinical anemic tumor models, rEpo has been shown to restore radiosensitivity, increase tumor growth delay (66–70), and increase cytotoxicity of chemotherapeutics (71). In a preclinical model, Blackwell et al. (72) found that systemic rEpo administration to nonanemic rats bearing mammary adenocarcinoma flank tumors improved tumor oxygenation independent of the effects on hemoglobin levels. Other studies have similarly shown a decrease in the hypoxic fraction of solid tumors with systemic rEpo treatment: one in an i.p. hemorrhagic ascites model of anemia in mice (73) and another in a total body irradiation anemia model in rats in which radiotherapy efficacy was also increased by rEpo administration (67). Epo alone was not associated with modulation of microvessel density or tumor growth rate in these studies.

The available rEpo preparations include epoetin-α, epoetin-β, and the longer-acting darbepoetin-α, and are effective in increasing hemoglobin levels, decreasing the need for red cell transfusions, and improving quality of life in cancer patients receiving therapy (1–3, 74). In several clinical trials, improvements in survival were suggested with rEpo administration to anemic cancer patients. In a retrospective study, significant improvements in response, control, and survival rates were observed in patients with squamous cell carcinomas of the oral cavity and oropharynx treated with chemoradiation and rEpo (75). Trends toward improved survival with rEpo treatment of anemic cancer patients were also reported in a randomized, double-blind, placebo-controlled trial of nonmyeloid malignancies (4) and a study of lung cancer patients receiving chemotherapy (74), although the latter was not sufficiently powered to evaluate survival. In contrast, two recent clinical trials reported adverse outcome associated with rEpo therapy of metastatic breast cancer patients undergoing chemotherapy (7) and head and neck cancer patients undergoing radiotherapy (6). The unfavorable survival rate in rEpo-treated patients was associated, at least in part, with disease progression in both trials. Additionally, rEpo administration seemed to be associated with an increased incidence of deep venous thrombosis in the breast cancer trial, as well as in a trial of concurrent chemoradiation in cervical cancer patients (76). Several design- and treatment-related issues have been pointed out in the breast and head-neck cancer trials, making their interpretation difficult. In contrast to the results of these trials, a recent meta-analysis provided suggestive, but inconclusive, evidence that rEpo may improve overall survival in anemic cancer patients (5). Further prospective, randomized, and controlled studies will be required to investigate the effects, if any, of rEpo therapy on disease progression and survival of cancer patients.

Epigenetics and Human Infectious Diseases

Hans Helmut Niller , Janos Minarovits , in Epigenetics in Human Disease , 2012

21.2.6 Uropathogenic Escherichia coli Infection Down-Regulates CDKN2A (p16 INK4A )

Coculture of uropathogenic E. coli with human uroepithelial cell lines strongly induced DNMT1 expression in comparison with non-pathogenic strains. In parallel, the tumor suppressor CDKN2A and the DNA repair gene MGMT were down-regulated, while a set of other genes (CDH1, MLH1, DAPK1, and TLR4) were not affected. Down-regulation of CDKN2A correlated to DNA methylation of its promoter. However, the MGMT gene was not methylated. Frequent UPEC infections might increase the risk for bladder cancer through increasing methylation of TSGs [84] .

Cancer Cell Division

Cancer cell division. Cancer cells divide even when the cells are not being 'told' to (no man standing on the switch).

When it comes to cell division, cancer cells break just about all the rules!

  • Cancer cells can divide without appropriate external signals.This is analogous to a car moving without having pressure applied to the gas pedal. An example would be the growth of a breast cancer cell without the need for estrogen, a normal growth factor. Some breast cancer cells actually lose the ability to respond to estrogen by turning off expression of the receptor for estrogen within the cell. These cells can still reproduce by bypassing the need for the external growth signal.
  • Cancer cells do not exhibit contact inhibition.While most cells can tell if they are being 'crowded' by nearby cells, cancer cells no longer respond to this stop signal. As shown above, the continued growth leads to the piling up of the cells and the formation of a tumor mass.
  • Cancer cells can divide without receiving the 'all clear' signal.While normal cells will stop division in the presence of genetic (DNA) damage, cancer cells will continue to divide. The results of this are 'daughter' cells that contain abnormal DNA or even abnormal numbers of chromosomes. These mutant cells are even more abnormal than the 'parent' cell. In this manner, cancer cells can evolve to become progressively more abnormal.

Continued cell division leads to the formation of tumors. The genetic instability that results from aberrant division contributes to the drug resistance seen in many cancers. Mutations in specific genes can alter the behavior of cells in a manner that leads to increased tumor growth or development.

More information on this topic may be found in Chapter 8 of The Biology of Cancer by Robert A. Weinberg.

Stem Cells

The second edition of Stem Cells: Scientific Facts and Fiction provides the non-stem cell expert with an understandable review of the history, current state of affairs, and facts and fiction of the promises of stem cells. Building on success of its award-winning preceding edition, the second edition features new chapters on embryonic and iPS cells and stem cells in veterinary science and medicine. It contains major revisions on cancer stem cells to include new culture models, additional interviews with leaders in progenitor cells, engineered eye tissue, and xeno organs from stem cells, as well as new information on "organs on chips" and adult progenitor cells.

In the past decades our understanding of stem cell biology has increased tremendously. Many types of stem cells have been discovered in tissues that everyone presumed were unable to regenerate in adults, the heart and the brain in particular. There is vast interest in stem cells from biologists and clinicians who see the potential for regenerative medicine and future treatments for chronic diseases like Parkinson's, diabetes, and spinal cord lesions, based on the use of stem cells and from entrepreneurs in biotechnology who expect new commercial applications ranging from drug discovery to transplantation therapies.

Tissue Culture, Cell Growth, and Analysis

Andrea L. Nestor , . David C. Allison , in Surgical Research , 2001

I. Introduction and Environment

Tissue culture is one of the most important research tools in cell biology. Nontransformed mammalian cells can be cultured for ∼35 generations, or to the “ Hayflick limit ,” before generalized senescence and cell death set in because of telomeric shortening. However, about 1 in 10 6 cells in such senescent cultures will spontaneously “transform” and become immortal. There seems to be no limit to the number of divisions possible for such spontaneously transformed lines, or for cell lines initially derived from malignant cells. Indeed, HeLa cells, derived from a human cervical cancer, have been in continuous passage for more than 50 years.

It is necessary to have the proper laboratory environment and equipment to perform tissue culture successfully. Ideally, the tissue culture area should be physically isolated from the general traffic flow of the laboratory. All precautions (see below) must be employed continuously to avoid contamination of the dedicated tissue culture work area, which should have at least three air changes per hour and contain the following equipment: a tissue culture incubator laminar flow high-efficiency particulate air (HEPA)-filtered hood centrifuge inverted phase microscope and a hemocytometer/light microscope and/or a Coulter counter for cell counting. Also, a refrigerator, autoclave, standard freezer (–4°C), ultralow freezer (–80°C), and a liquid nitrogen storage system (–130°C) for the long-term preservation of frozen cell lines should be available ( 1 ).

Henrietta Lacks’ ‘Immortal’ Cells

Medical researchers use laboratory-grown human cells to learn the intricacies of how cells work and test theories about the causes and treatment of diseases. The cell lines they need are “immortal”—they can grow indefinitely, be frozen for decades, divided into different batches and shared among scientists. In 1951, a scientist at Johns Hopkins Hospital in Baltimore, Maryland, created the first immortal human cell line with a tissue sample taken from a young black woman with cervical cancer. Those cells, called HeLa cells, quickly became invaluable to medical research—though their donor remained a mystery for decades. In her new book, The Immortal Life of Henrietta Lacks, journalist Rebecca Skloot tracks down the story of the source of the amazing HeLa cells, Henrietta Lacks, and documents the cell line's impact on both modern medicine and the Lacks family.

Related Content

Who was Henrietta Lacks?
She was a black tobacco farmer from southern Virginia who got cervical cancer when she was 30. A doctor at Johns Hopkins took a piece of her tumor without telling her and sent it down the hall to scientists there who had been trying to grow tissues in culture for decades without success. No one knows why, but her cells never died.

Why are her cells so important?
Henrietta’s cells were the first immortal human cells ever grown in culture. They were essential to developing the polio vaccine. They went up in the first space missions to see what would happen to cells in zero gravity. Many scientific landmarks since then have used her cells, including cloning, gene mapping and in vitro fertilization.

There has been a lot of confusion over the years about the source of HeLa cells. Why?
When the cells were taken, they were given the code name HeLa, for the first two letters in Henrietta and Lacks. Today, anonymizing samples is a very important part of doing research on cells. But that wasn’t something doctors worried about much in the 1950s, so they weren’t terribly careful about her identity. When some members of the press got close to finding Henrietta’s family, the researcher who’d grown the cells made up a pseudonym—Helen Lane—to throw the media off track. Other pseudonyms, like Helen Larsen, eventually showed up, too. Her real name didn’t really leak out into the world until the 1970s.

How did you first get interested in this story?
I first learned about Henrietta in 1988. I was 16 and a student in a community college biology class. Everybody learns about these cells in basic biology, but what was unique about my situation was that my teacher actually knew Henrietta’s real name and that she was black. But that’s all he knew. The moment I heard about her, I became obsessed: Did she have any kids? What do they think about part of their mother being alive all these years after she died? Years later, when I started being interested in writing, one of the first stories I imagined myself writing was hers. But it wasn’t until I went to grad school that I thought about trying to track down her family.

A HeLa cancer cell dividing. (© Dr. Thomas Deerinck / Visuals Unlimited / Corbis) The metaphase stage of a human HeLa cell division. (© Dr. Richard Kessel / Dr. Gene Shih / Visuals Unlimited / Corbis) Subspecies of HeLa cells have evolved in labs and some feel that the cell line is no longer human, but a new microbial life form. These cells are shown in green the cytoplasm is red and structures within the cytoplasm are blue. (© Nancy Kedersha / Science Faction / Corbis) The prophase stage of mitosis in the division of these human HeLa cells. (© Dr. Richard Kessel / Dr. Gene Shih / Visuals Unlimited / Corbis) This fluorescence micrograph of a HeLa cell shows the cytoskeletal microfilaments in red and nuclei stain with Hoechst in blue. (© Visuals Unlimited / Corbis)

How did you win the trust of Henrietta’s family?
Part of it was that I just wouldn’t go away and was determined to tell the story. It took almost a year even to convince Henrietta’s daughter, Deborah, to talk to me. I knew she was desperate to learn about her mother. So when I started doing my own research, I’d tell her everything I found. I went down to Clover, Virginia, where Henrietta was raised, and tracked down her cousins, then called Deborah and left these stories about Henrietta on her voice mail. Because part of what I was trying to convey to her was I wasn’t hiding anything, that we could learn about her mother together. After a year, finally she said, fine, let’s do this thing.

When did her family find out about Henrietta’s cells?
Twenty-five years after Henrietta died, a scientist discovered that many cell cultures thought to be from other tissue types, including breast and prostate cells, were in fact HeLa cells. It turned out that HeLa cells could float on dust particles in the air and travel on unwashed hands and contaminate other cultures. It became an enormous controversy. In the midst of that, one group of scientists tracked down Henrietta’s relatives to take some samples with hopes that they could use the family’s DNA to make a map of Henrietta’s genes so they could tell which cell cultures were HeLa and which weren’t, to begin straightening out the contamination problem.

So a postdoc called Henrietta’s husband one day. But he had a third-grade education and didn’t even know what a cell was. The way he understood the phone call was: “We’ve got your wife. She’s alive in a laboratory. We’ve been doing research on her for the last 25 years. And now we have to test your kids to see if they have cancer.” Which wasn’t what the researcher said at all. The scientists didn’t know that the family didn’t understand. From that point on, though, the family got sucked into this world of research they didn’t understand, and the cells, in a sense, took over their lives.

How did they do that?
This was most true for Henrietta’s daughter. Deborah never knew her mother she was an infant when Henrietta died. She had always wanted to know who her mother was but no one ever talked about Henrietta. So when Deborah found out that this part of her mother was still alive she became desperate to understand what that meant: Did it hurt her mother when scientists injected her cells with viruses and toxins? Had scientists cloned her mother? And could those cells help scientists tell her about her mother, like what her favorite color was and if she liked to dance.

Deborah’s brothers, though, didn’t think much about the cells until they found out there was money involved. HeLa cells were the first human biological materials ever bought and sold, which helped launch a multi-billion-dollar industry. When Deborah’s brothers found out that people were selling vials of their mother’s cells, and that the family didn’t get any of the resulting money, they got very angry. Henrietta’s family has lived in poverty most of their lives, and many of them can’t afford health insurance. One of her sons was homeless and living on the streets of Baltimore. So the family launched a campaign to get some of what they felt they were owed financially. It consumed their lives in that way.

These HeLa cells were stained with special dyes that highlight specific parts of each cell. The DNA in the nucleus is yellow, the actin filaments are light blue and the mitochondria—the cell's power generators—are pink. (© Omar Quintero) Henrietta Lacks' cells were essential in developing the polio vaccine and were used in scientific landmarks such as cloning, gene mapping and in vitro fertilization. (Courtesy of the Lacks family) Margaret Gey and Minnie, a lab technician, in the Gey lab at Johns Hopkins, circa 1951. (Courtesy of Mary Kubicek) In The Immortal Life of Henrietta Lacks, journalist Rebecca Skloot tracks down the story of the source of the amazing HeLa cells. (Courtesy of Random House, Inc.) Skloot first learned about Henrietta in 1988 from a community college biology teacher. (Courtesy of Random House, Inc.)

What are the lessons from this book?
For scientists, one of the lessons is that there are human beings behind every biological sample used in the laboratory. So much of science today revolves around using human biological tissue of some kind. For scientists, cells are often just like tubes or fruit flies—they’re just inanimate tools that are always there in the lab. The people behind those samples often have their own thoughts and feelings about what should happen to their tissues, but they’re usually left out of the equation.

And for the rest of us?
The story of HeLa cells and what happened with Henrietta has often been held up as an example of a racist white scientist doing something malicious to a black woman. But that’s not accurate. The real story is much more subtle and complicated. What is very true about science is that there are human beings behind it and sometimes even with the best of intentions things go wrong.

One of the things I don’t want people to take from the story is the idea that tissue culture is bad. So much of medicine today depends on tissue culture. HIV tests, many basic drugs, all of our vaccines—we would have none of that if it wasn’t for scientists collecting cells from people and growing them. And the need for these cells is going to get greater, not less. Instead of saying we don’t want that to happen, we just need to look at how it can happen in a way that everyone is OK with.


In this paper we inferred a mathematical model of tumor spheroid growth for the non-small cell lung cancer cell line SK-MES-1 from image data of growing tumor spheroids. Cell nuclei, proliferating cells, extra-cellular matrix and dying cells (by either necrosis or apoptosis) were labeled at different points in time and under different oxygen and glucose medium concentrations. The model was built by an iterative procedure, which we propose as a general template for modeling tissue organization processes. We started by developing a minimal model for one growth condition only, then stepwise extending this model by further mechanisms whenever the previous simpler model turned out to be insufficient to reproduce the experimental observations for an additional growth condition. Before adding a new mechanism to an existing model version we verified by extensive computer simulations (usually hundreds of runs), that within the parameter range for each parameter of the existing model no satisfying agreement between model and data could be achieved. Minimal is here to be understood as sufficient to explain the data and containing as least mechanisms as possible, whereby the building blocks of the model were chosen from those mechanisms that have already been described somewhere for any cell population. A similar iterative strategy was pursued for liver regeneration after drug induced damage predicting a previously unrecognized and subsequently validated order mechanisms [65].

We studied four different combinations of glucose and oxygen in the medium. To explain the growth kinetics, the proliferation, ECM, and cell death for the condition with high glucose and high oxygen medium concentration ([G] = 25mM, [O] = 0.28mM), the second with intermediate concentration of glucose and high oxygen concentration ([G] = 5mM, [O] = 0.28mM), we needed to assume that the cell cycle progression is possible only above a critical local production rate of ATP (= mM/h). A second necessary condition was, that the local density of extra-cellular matrix had to be higher than a critical value (0.003). This is in accordance to literature, where dependence of cancer progression on the ECM has been shown for skin cancer [66], breast cancer [67] and NSCLC [68], where Collagen IV can regulate crucial cell signaling. If both conditions (enough ATP and ECM) were fulfilled, cells could reenter the cell cycle after a cell division. Here, cells, which were closer to the spheroid surface and thus needed less energy in order to expand, had an increasing chance to continue proliferation and not to become quiescent. Interestingly the decision whether a cell in a certain condition became quiescent, had to be stochastic. This introduced some heterogeneity in subsets of cells in the same conditions. A deterministic scenario could not have explained the smooth transition from proliferating to quiescent zones.

The production rate of ATP depended on the local oxygen and glucose concentrations. Thereby, the ratio between both dictates to which extent a cell is in the aerobic Krebs cycle or the anaerobic lactate fermentation. Warburg stated in [69] that all cancer cells suffer from an injured respiration and thus have an exclusively anaerobic metabolism. In opposition, Zu and Guppy [70] disproved this hypothesis due to the lack of evidence and rather claimed the metabolism in cancer cells to be functional, but mainly glycolytic due to hypoxia. Here we come to a partially different conclusion: if cells are sufficiently supplied with glucose (independent from the oxygen supply), the metabolism will remain glycolytic (90%), and only if the glucose supply is getting short, the metabolism will favor the aerobic Krebs cycle (see Fig 8). Besides lactate acidity (> 20mM), the depletion of carbon sources to maintain a critical ATP production (= 900mM/h) and not hypoxia were the main reasons of death. The latter was also recently suggested by Kasinskas et al. [71], while, in contrast to our assumptions, they excluded lactate as source of acidity and instead assumed it to be an important secondary metabolic resource. However, either growth adverse or death promoting effects were described for high lactate concentrations [59]. So here further clarification of the dominating role of lactate would be necessary. The functional forms of the oxygen and glucose consumption rates were inferred from experimental findings of Freyer, Sutherland and co-workers in EMT6/Ro cells. The lactate and ATP production rates were then directly derived from those rates by the single assumption that cells transform the consumed glucose in an optimal way with respect to ATP output. For wide ranges of glucose and oxygen concentrations the ATP production rates remain stable between 80…130 × 10 −17 mol/cell/s or 1000…1700mM/h respectively, assuming a reference cell volume of 2700μm. In literature values can be found between 4.6…15.3 × 10 −17 mol/cell/s ([72–75]). The difference could be either due to differences in energy needs between different cell types, or to the model simplification that glucose in our model is exclusively used for metabolism.

Interestingly and importantly, the model, despite only having been calibrated with two of the four growth conditions, were subsequently able to correctly and quantitatively predict the growth phase of the other two growth conditions ([G] = 1mM, [O] = 0.28mM and [G] = 25mM, [O] = 0.07mM, respectively). This indicates that the model did capture the functionalities necessary to explain the data for different glucose and oxygen conditions. To further permit independent validation of our model, we performed additional simulations for other glucose and oxygen medium concentrations (Fig S12 in S1 Document).

However, all growth curves showed saturation and partially even shrinkage after some time. The saturation phase could be largely captured by adding the potential effect of a waste produce being released in the extracellular space from cells undergoing lysis. Shrinkage could be added if dying cells at the border detach and enter the growth medium however, we did not consider this process, as it was not observed in the experiments (for example, for A549 cells, another NSLC cell line, a massive detachment of cells from the spheroid could be observed in the experiments). Interestingly, model simulations with a lysis rate of 0.35/h, a typical value in-vivo, turned out to be incompatible with the in-vitro data. A lysis rate of a few hours as observed in-vivo would lead to a very fast removal of dying cells and thus almost no dead cells in the tumor center, in sharp contast to the in-vitro experiments. We obtained a much smaller value of about 0.01/h by comparison of model simulation results and the spatial cell death and proliferation profiles i.e., only such a small lysis rate permits the occurrence of a “necrotic core” as observed in the in-vitro experiments. For such a low lysis rate we found that the apoptosis—if present—would need to be very slow, as it affects also cells in the viable rim in order to agree with the experimental observation of only very little dead cells in the viable rim. For this reason, apoptosis could be neglected in explaining the experimental results in this paper. The small value of the lysis rate, even though surprising on a first view, may be explained by noticing that stromal cells (such as e.g. macrophages) digesting dead cells are not present in-vitro. Hence lysis might be expected to be slower in-vitro than in-vivo.

Contact inhibition seems to be a crucial element. Suppressing contact inhibition with varying combinations of the other mechanisms in each case leads to complete failure of match between data and model simulations (see Fig S6 in S1 Document, where the parameters of model 4 has been used). This observation supports the view expressed previously in the paper that a mechanical growth inhibition plays an important role in multicellular spheroids.

We moreover tested the possibility that cells may actively migrate towards the necrotic zone by necrotaxis (Figs S9, S10 in S1 Document). As to keep a sufficiently large necrotic core as experimentally observed the lysis rate had to be small, significant migration could not be observed. On the other hand, if the lysis rate was chosen large, then significant migration of cells could be observed but the necrotic core was too small, as cells in the center were too quickly eliminated by lysis. In the latter case, the necrotic core with increasing migration rate became smaller (Figs S9, S10 in S1 Document). We concluded that migration driven by morphogens towards the central necrosis in SK-MES-1 cells is small.

Interestingly the final model emerging from this stepwise, image-guided inference strategy closely resembles the hypothesis on growth control of MCTS by growth promoters (GP), growth inhibitors (GI), viability promoters (VP) and inhibitors (VI) (Fig 11).