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

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: (1) Invasive cells that produce an acidic environment via upregulated aerobic glycolysis, and (2) Non-invasive 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 non-invasive, 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 micro-environmental pHe could invert the cost/benefit ratio of the C2 strategy and select for C3 cells. In vivo, 200mM 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 co-injected 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.

Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR

“…To do so, we used tumor specimens available from previous studies (Estrella et al, 2013; Ibrahim-Hashim et al, 2017) in which tumor xenografted mice drank either tap water or water supplemented with sodium bicarbonate, a method shown in these studies and by others (Gallagher et al., 2008) to raise intratumoral pH and, notably, intracellular pH (Raghunand et al, 1999). We assessed phosphorylated S6 by immunohistochemistry as a measure of mTORCI signaling.…”

“…In two tumor models (colon cancer HCT116, breast cancer MCF7), sodium bicarbonate administration significantly elevated mTORCI signaling (Figures 7K, S7L), consistent with in vitro corollary work with these cell lines showing acid sufficient to inhibit mTORCI signaling and additional bicarbonate (highly buffered media) able to blunt HIF-mediated mTORCI suppression (Figures S7J-K). Importantly, mTORCI activation did not stem from bicarbonate stimulating proliferation, as bicarbonate either slowed (HCT 116) or did not affect (MCF7) tumor growth rates (Estrella et al, 2013; Ibrahim-Hashim et al, 2017). A third model (breast cancer MDA-MB-231) revealed high baseline tumor mTORC1 activity not further augmented by bicarbonate therapy (Figure S7M).…”

“…Tumor tissue specimens were obtained from two previously conducted studies (Estrella et al, 2013; Ibrahim-Hashim et al, 2017) in which the drinking water of mice bearing xenograft tumors was either supplemented with bicarbonate to raise intratumoral pH or not supplemented (“tap” water). These tissues were then queried for the current study for the impact of bicarbonate therapy on tumor mTORC1 signaling.…”

“…For MDA-MB-231 and MCF7 tumor formation, 10 million cells were injected into cleared mammary fat pads as described previously (Ibrahim-Hashim et al, 2017). One week prior to cell injection, an estrogen pellet (0.72 mg slow release, Innovative Research of America) was implanted to allow growth of ER-positive MCF7 tumors.…”

Connecting Cancer to Its Causes Requires Incorporation of Effects on Tissue Microenvironments

“…A more effective strategy for cancer prevention could be to alter tissue landscapes in order to limit selection for the inevitable oncogenic mutations. Therapeutic strategies could be used to alter the selective value of cancer phenotypes by increasing the fitness of more benign phenotypes and/or disfavoring malignant genotypes through modulating tissue parameters, as has been shown for pH (27), oxygen levels (28), cytokines (5,29), and the presence of senescent cells (30). In each case, treatments engendering a tissue microenvironment that better approached that of a young healthy individual, such as by ameliorating inflammation, reduced the selective value of malignant or aggressive genotypes and thus impaired cancer initiation and progression.…”