Abstract 4117: Defective quorum sensing in Ph+ ALL and identification of diffusible factors secreted by cells growing at high cell density.

Abstract

Quorum sensing (QS) is a generic term describing cell-cell communication and collective decision making by bacteria and social insects to regulate expression of specific genes controlling cell density and other properties in response to changes in nutrient supply or environment. QS also has a role in higher organisms in maintaining homeostasis, regulation of the immune system and behavior of cancer cell populations. CML is a classic example of a tumor which without effective therapy consistently undergoes malignant progression, replacing the chronic phase (CP) cells with undifferentiated blast cells. Like most other types of acute leukemia including Ph+ ALL, the CML blasts proliferate slower than either normal or CP CML stem/progenitor (S/P) cells and individual blasts have greatly reduced proliferative potential and cloning ability suggesting the leukemic blasts achieve dominance by functioning as an aberrant ecosystem subject to different regulatory mechanisms than normal or CP CML S/P cells. To see if QS is involved we selected a p190 bcr-abl driven pre-B cell line (ALL3) derived from the pleural fluid of a patient dying of Ph+ ALL. Unlike other established human or murine Bcr-Abl driven lines, ALL3 cells don't form colonies or grow at low cell densities (LD) but grow progressively faster at increasing densities (HD). The cells are unresponsive to any known cytokines or combinations at any density. ALL3 cells at HD in upper inserts of transwells stimulate growth of LD cells in lower wells and LD ALL3 are also stimulated to grow by diffusible factors in the supernate (SN) of HD cells. Unstimulated LD ALL3 maintain fair viability (Trypan blue exclusion) for several days, and cells are also poised to begin proliferating; almost 98-100% are Ki-67+ and ∼40% incorporate BrdU, but an increasing number undergo apoptosis and only a minority regrow with addition of HD SN. The stimulatory factor(s) fail to stimulate other HD ALL3 cells, suggesting novel sensory circuits are activated upon population depletion or lack of close contact. LD ALL3 cells are also stimulated by SNs of enriched CD34+ cells at HD from normal PB, cord blood or CP CML but not by less enriched MNCs; it is not yet known if these stimulatory effects are due to the same or different factor(s). Our first objective is to identify the stimulatory protein(s) in the secretomes of HD ALL3 and other cells. The HD SN is stable upon storage at 4°C or -80°C with no loss of activity. Using cutoff filters all stimulatory activity is in the >30kDa Fx and with anion exchange chromatography activity is concentrated in 1 or 2 cationic flow-through sub-fractions. We are currently working with several proteomic experts to identify and characterize the stimulatory factors and the novel pathways and regulatory mechanisms involved. Our ultimate goal is to define the aberrant circuitries and find vulnerable targets for specific therapies of leukemias and other cancers. Citation Format: Sapan J. Patel, Dao Su, Bayard Clarkson. Defective quorum sensing in Ph+ ALL and identification of diffusible factors secreted by cells growing at high cell density. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4117. doi:10.1158/1538-7445.AM2013-4117

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell–cell communication

“…A newly established cell line freshly obtained from the leukemic cells growing as ascitic cells in the pleural effusion of a terminally ill patient with Ph+ acute lymphoblastic leukemia (ALL3) is used in this work. ALL3 is a clonal proliferation of p190 BCR-ABL transformed pre B cells that arise in the bone marrow [30,34]. We observed that there is a significant difference in the growth rate of ALL3 cells at low (LD) and high starting cell densities (HD).…”

“…However, the influence of cell-cell communication on biomechanical properties of cells has not been systematically studied. At the same time, it is known that the collective behavior is an important feature and it is involved in regulating many biological processes such as cell migration, stem cell maintenance, growth of proper organ size, immune system regulation, hematopoiesis, homeostasis and regeneration [27][28][29][30][31][32][33][34]. Individual cells utilize 'autocrine' and/or 'paracrine' factors to coordinate these beneficial collective behaviors.…”

“…Individual cells utilize 'autocrine' and/or 'paracrine' factors to coordinate these beneficial collective behaviors. Even prokaryotic cells utilize these quorum-sensing (QS) molecules to 'count' their population numbers to determine whether the conditions are suitable to perform specific tasks including complex behaviors, such as formation of complex biofilms, antibiotic production, motility, sporulation, virulence, competence, conjugation and symbiosis [30,[34][35][36][37][38]. Cancer cell populations also function collectively to initiate and maintain abnormal cell proliferation, permit invasion and metastasis, avoid inhibition by the immune system, develop therapeutic resistance, and metabolic reprogramming [39,40].…”

“…Cancer cell populations also function collectively to initiate and maintain abnormal cell proliferation, permit invasion and metastasis, avoid inhibition by the immune system, develop therapeutic resistance, and metabolic reprogramming [39,40]. The underlying biochemical and biological QS mechanisms responsible for the deviant behavior of cancer cell populations are still poorly understood [30,34,[41][42][43].…”

“…Thus, cell proximity as well as overall cell density are important. Labeling studies with proliferation markers Ki67, BrdU or EdU showed that the LD ALL3 cells were poised to begin proliferating but could not do so without being triggered by supernate (HDSN) from ALL3 cells or some other normal or leukemic cells growing at HD [30,34].…”