Nutritional Niches of Cancer Therapy-Induced Senescent Cells

Abstract

Therapy-induced senescence (TIS) is a state of stable proliferative arrest of both normal and neoplastic cells that is triggered by exposure to anticancer treatments. TIS cells acquire a senescence-associated secretory phenotype (SASP), which is pro-inflammatory and actively promotes tumor relapse and adverse side-effects in patients. Here, we hypothesized that TIS cells adapt their scavenging and catabolic ability to overcome the nutritional constraints in their microenvironmental niches. We used a panel of mechanistically-diverse TIS triggers (i.e., bleomycin, doxorubicin, alisertib, and palbociclib) and Biolog Phenotype MicroArrays to identify (among 190 different carbon and nitrogen sources) candidate metabolites that support the survival of TIS cells in limiting nutrient conditions. We provide evidence of distinguishable TIS-associated nutrient consumption profiles involving a core set of shared (e.g., glutamine) and unique (e.g., glucose-1-phosphate, inosine, and uridine) nutritional sources after diverse senescence-inducing interventions. We also observed a trend for an inverse correlation between the intensity of the pro-inflammatory SASP provoked by different TIS agents and diversity of compensatory nutritional niches utilizable by senescent cells. These findings support the detailed exploration of the nutritional niche as a new metabolic dimension to understand and target TIS in cancer.

Keywords: cancer; glutamine; metabolism; miR146a; nutrition; senescence.

Figure 1

Metabolic fingerprinting of therapy-induced senescence: an experimental approach. (A) A549 cells were cultured for 7 days with bleomycin (20 μmol/L), alisertib (0.5 μmol/L), doxorubicin (50 nmol/L), and palbociclib (5 μmol/L). Top: representative images of SA-β-gal staining from three independent experiments. Scale bar: 200 μm. Bottom: representative images from 6-well plates of 10-day clonogenic survival analyses of A549 cells previously cultured for 7 days with therapy-induced senescence agents. (B) Schematic representation of metabolite utilization analysis workflow in proliferative versus TIS cells using the Phenotype MicroArrays PM-M1 and PM-M2.

Figure 2

Substrate utilization patterns of therapy-induced senescence cells. (A) Representative images of paired proliferative/TIS cells assayed in PM-M1 and PM-M2 plates. Negative control wells (blue boxes) have no substrate. Wells containing D-glucose (red boxes) served as positive controls. (B) Flower model Venn diagrams showing higher (left) or lower (right) substrate utilization in each type of TIS cell. (C) Analysis of mitochondrial oxidation of glucose and glutamine in proliferative and TIS cells using the Agilent Seahorse XF Mito Fuel Flex kit.

Figure 3

Nutritional rescue of glucose deprivation in therapy-induced senescence cells. Flower model Venn diagrams showing shared and unique metabolic substrates capable of rescuing at least (A) one-fifth or (B) one-quarter of the bioenergetic capacity of glucose in TIS cells.

Figure 4

Activation levels of the negative-feedback regulator of the SASP response, miR-146a, and diversity of nutritional rescue of glucose deprivation in therapy-induced senescence cells. (A) The miR146a-EGFP reporter detects senescence in A549 cells. The SASP-responsive miR146a-EGFR reporter was transfected into A549 cells and TIS was induced through treatment with bleomycin, alisertib, doxorubicin, and palbociclib. EGFP fluorescence was measured using either IncuCyte Zoom or flow cytometry. Acquisition and analysis of images was carried out using fully integrated algorithms in an IncuCyte S3 analysis system. (B) Left: kinetic plots of SASP-driven miR146a-EGFP reporter expression following senescence induction with bleomycin, alisertib, doxorubicin, and palbociclib. Right: correlation between SASP intensity and number of metabolites (as calculated in Figure 3A,B) circumventing glucose-deprived conditions following senescence induction with bleomycin, alisertib, doxorubicin, and palbociclib.

Figure 5

Nutritional fingerprints of therapy-induced senescent cells: linking senescent phenotypes to metabolic niches. Cells in a TIS state share several common features, such as changes in morphology, increase in β-galactosidase activity, and activation of an inflammatory response. Understanding whether all types of senescent cells induced by exogenous therapeutic stresses are identical or heterogeneous in terms of their metabolic needs might aid in achieving the goal of selectively eliminating the deleterious effects of TIS cells. The recognition of inter-TIS heterogeneity in terms of scavenging and catabolic ability contingent on nutrient availability might help to molecularly understand and therapeutically exploit TIS phenotype-associated “communities” linked to contextual micro-environmental nutritional cues in complex cancer and normal tissues.