Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 1;77(9):2242-2254.
doi: 10.1158/0008-5472.CAN-16-2844. Epub 2017 Mar 1.

Defining Cancer Subpopulations by Adaptive Strategies Rather Than Molecular Properties Provides Novel Insights Into Intratumoral Evolution

Affiliations
Free PMC article

Defining Cancer Subpopulations by Adaptive Strategies Rather Than Molecular Properties Provides Novel Insights Into Intratumoral Evolution

Arig Ibrahim-Hashim et al. Cancer Res. .
Free PMC article

Abstract

Ongoing intratumoral evolution is apparent in molecular variations among cancer cells from different regions of the same tumor, but genetic data alone provide little insight into environmental selection forces and cellular phenotypic adaptations that govern the underlying Darwinian dynamics. In three spontaneous murine cancers (prostate cancers in TRAMP and PTEN mice, pancreatic cancer in KPC mice), we identified two subpopulations with distinct niche construction adaptive strategies that remained stable in culture: (i) invasive cells that produce an acidic environment via upregulated aerobic glycolysis; and (ii) noninvasive cells that were angiogenic and metabolically near-normal. Darwinian interactions of these subpopulations were investigated in TRAMP prostate cancers. Computer simulations demonstrated invasive, acid-producing (C2) cells maintain a fitness advantage over noninvasive, angiogenic (C3) cells by promoting invasion and reducing efficacy of immune response. Immunohistochemical analysis of untreated tumors confirmed that C2 cells were invariably more abundant than C3 cells. However, the C2 adaptive strategy phenotype incurred a significant cost due to inefficient energy production (i.e., aerobic glycolysis) and depletion of resources for adaptations to an acidic environment. Mathematical model simulations predicted that small perturbations of the microenvironmental extracellular pH (pHe) could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200 mmol/L NaHCO3 added to the drinking water of 4-week-old TRAMP mice increased the intraprostatic pHe by 0.2 units and promoted proliferation of noninvasive C3 cells, which remained confined within the ducts so that primary cancer did not develop. A 0.2 pHe increase in established tumors increased the fraction of C3 cells and signficantly diminished growth of primary and metastatic tumors. In an experimental tumor construct, MCF7 and MDA-MB-231 breast cancer cells were coinjected into the mammary fat pad of SCID mice. C2-like MDA-MB-231 cells dominated in untreated animals, but C3-like MCF7 cells were selected and tumor growth slowed when intratumoral pHe was increased. Overall, our data support the use of mathematical modeling of intratumoral Darwinian interactions of environmental selection forces and cancer cell adaptive strategies. These models allow the tumor to be steered into a less invasive pathway through the application of small but selective biological force. Cancer Res; 77(9); 2242-54. ©2017 AACR.

Conflict of interest statement

Conflict of interest All authors have no conflict of interest to disclose

Figures

Figure 1
Figure 1
A–D. In vitro profiling of the tumorigenic TRAMP-C2 and non-tumorigenic TRAMP-C3 cell lines, A. Metabolic flux analysis of Extracellular Acidification Rate (ECAR) was measured in real-time using a Seahorse XFe-96 analyzer. C2 cells exhibit increased glycolysis, compared to C3 cell line; *p=0.0274. Glycolytic capacity, was higher in the C2 cells; **p=0.001. B. The basal Oxygen Consumption Rates (OCR) of the two cell lines was not different. The C3 cells have a slight, albeit, significant increase in respiratory capacity; **p=0.0082. C. In vitro motility which measured by impedance that is caused by migration in both cell lines and recorded by the xCELLigence Real-Time Cell Analyzer (RTCA) instrument, C2 cells had high migration, and C3 line was not motile; ***p=0.0007.D. Invasiveness comparison between C2- and C3- cells measured by increases in impedance following trans-well migration assay, the C2 cells were more invasive; **p=0.0015. A Mann Whitney statistical test was used to show that the two time series curves are from different populations. Both migration and invasion were normalized to proliferation rate.
Figure 2
Figure 2
Effect of bicarbonate on perfusion in TRAMP. (A–C), Tumor vasculature (perfusion and permeability) was assessed using gadolinium (Gd)–based dynamic contrast enhanced (DCE)-MRI, A. Representative Images of DCE-MRI images in coronal plane of the TRAMP prostate tumor in Tap group and 200 mM bicarbonate group are shown. Images were obtained at time 0 (prior to Gd injections), and at 4, 7, and 10 minutes post Gd injections. The color scale represents incremental increase in signal intensity in the prostate. B. Data analysis for initial area under the curve (AUC) of the signal intensity was performed using Matlab, showing higher blood flow in the 200 mM bicarbonate treated group (****P<0.0001). C. Statistical analyses of enhancement at 10 minutes, showing mean signal intensity ± SD, as well as the skewness of the histogram of enhancement values ± SD. The mean values indicate significantly increased enhancement (p=0.001), in the treated, compared to the Tap controls. Further, the enhancing pixels in the tap group were more normally distributed, compared to the bicarbonate group (p=0.03), which we interpret as recruitment of new vasculature that is skewed towards higher perfusion values. Two-tailed Student’s t-tests were used to calculate statistical significance.
Figure 3
Figure 3
Results from a multiscale mathematical model of tumor growth for different starting times and doses of sodium bicarbonate. A Modeled interactions between microenvironmental components, in yellow (vasculature, oxygen, acidosis) and tumor cell phenotypes (aerobic (green), acid-resistant (blue), glycolytic (red), glycolytic and acid-resistant (pink)). B. Cell life-cycle flowchart that every tumor cell obeys, showing input parameters of oxygen, pH, ATP and space, and resulting cell decisions of quiescence, death, or proliferation. C. Phenotype space and D. Physical space is shown at t=200 days for the following treatment conditions: 1) low dose bicarbonate given early (t=40d), 2) high dose given early, 3) low dose given late (t=150d), 4) high dose given late, 5) no treatment. Initial conditions for each simulation are identical and shown as inset S in each figure D. For each simulation, the colors of the tumor cells correspond to their position in phenotype space, (panel C) where the horizontal axis is the level of glycolytic capacity and the vertical axis is the amount of acid resistance. Estimated regions corresponding to C2 (blue/magenta) and C3 (green) phenotypes are indicated. E. Corresponding growth curves, with tumor size (area in mm2) and time in days. The simulation windows (D) are approximately 4.5 by 4.5 mm in size. (See Movie provided as a supplemental Figure).
Figure 4
Figure 4
Reduction of tumor growth in TRAMP model. A. In vivo measurements of the pH of the prostate in tap, 200mM bicarbonate-treated groups, and 400mM bicarbonate treated group (n=3 for each cohort) were obtained immediately prior to euthanasia. pH was measured using single-barrel pH microelectrode, MI-419 (Microelectrode, Inc., Londonderry NH). The results indicate a statistically significant increase in pH in the bicarbonate treated animals compared to the tap animals; * p=0.015. * p =0.04. Mean ± standard error of the mean (SEM) are shown. B. Prostate volume measurements of different treatments; non-transgenic (n=5), TAP (n=7), late start 200mM bicarbonate treatment (n=5), early treatment at 200mM bicarbonate (n=5) and late treated with 400mM bicarbonate (n=7) cohorts obtained through US imaging. Data show that mice treated with higher dose (400mM sodium bicarbonate) at 4 weeks of age maintained reduced prostate volumes throughout study (*p=0.016), while the late treated mice (started at 10 weeks) only showed reduced volume at 24 weeks (*p=0.03). C. Histological Quantitative analysis of prostate tumors showing percent pixels associated with benign and malignant phenotypes in prostate tumors of different treatment cohorts. These show significant differences in the percent of benign and malignant tumors observed in the 4 week old treated mice and late treated with increased does compared to other groups; * p=0.017, and *p=0.03 respectively. A two-tailed Student’s t-test was used to calculate statistical significance.
Figure 5
Figure 5
Reduction of tumor metastasis in TRAMP model. A. Simulated growth of metastases under untreated (green curve), low dose (red) and high dose (blue) buffer therapy. The horizontal axis represents the phenotype of the initial metastatic seed cell. Seeds were selected from positions along the diagonal white line in the phenotype plot of Figure 4 (panel C), where the value of 0 in this plot corresponds to position S (normal phenotype) and the value of 1 corresponds to the most glycolytic and acid resistant phenotype, the upper right corner of Figure 4A. For reference, the color gradient represents the seed phenotypes, with C3 and C2 cell types as marked. The vertical axis shows the final size of the metastasis in mm2, measured at t=120d after the metastatic cell was seeded. The buffer therapy was administered from t=0 until the end of the simulation. Each point is the average of 4 runs. High dose therapy suppresses metastases of all seed types; aggressive C2 seeds still grow under low dose therapy. B. Lungs and liver metastasis images (A respectively from three different treatment cohorts. In Tap cohort we observed metastasis, while in bicarbonate treated cohorts, minimal metastases were observed. Black arrow is normal tissue, white arrow is metastasis. C. Quantification of metastasis in lungs and Liver of three different cohorts, showing significant decrease in lung metastasis (**p= 0.001) in early treatment and (*p=0.03) in late treatment groups compared to tap. For liver, *p= 0.01 for early treatment and *p=0.02 for late treatment compared to tap. Mean ± standard error of the mean (SEM) are shown. A two-tailed Student’s t-test was used to calculate statistical significance.
Figure 6
Figure 6
A. Top images are H&E images of representative tumors of each cell line and mix (MDA-MB23 and MCF7, 1:1) under different treatments. Lower images are ER staining of consecutive slices of the same tumors as in H&E. B. Analysis of ER expression in all the tumors used in this experiment. Note how ER expression is bigger (p-value = 0.0001) in those tumors (composed by a mixture of MDA-MB231 and MCF7, 1:1) treated with bicarbonate 400 mM than in those under Tap water. C. MRI volumetric data of all the tumors used in this experiment. Data are represented as fold of change in tumor volume versus days after tumor injection. Mean ± standard error of the mean (SEM) are shown. A two-tailed Student’s t-test was used to calculate statistical significance.
Figure 7
Figure 7
Model for manipulation of niche construction evolutionary strategy, Our hypotheses is that, tumor consist of two distinct subpopulations of cells, highly glycolytic, acid producing cells (red cells), and non-glycolytic, non-acid producing cells (blue cells). Our model predicts that small perturbations in extracellular pH (pHe) could induce a population phase transition favoring the non-acid-producing, non-invasive cancer populations’

Similar articles

See all similar articles

Cited by 27 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback