Cellular Packing Density Research Articles

Morphophenotypic classification of tumor organoids as an indicator of drug exposure and penetration potential.

The dynamics of tumor progression is driven by multiple factors, which can be exogenous to the tumor (microenvironment) or intrinsic (genetic, epigenetic or due to intercellular interactions). While tumor heterogeneity has been extensively studied on the level of cell genetic profiles or cellular composition, tumor morphological diversity has not been given as much attention. The limited analysis of tumor morphophenotypes may be attributed to the lack of accurate models, both experimental and computational, capable of capturing changes in tumor morphology with fine levels of spatial detail. Using a three-dimensional, agent-based, lattice-free computational model, we generated a library of multicellular tumor organoids, the experimental analogues of in vivo tumors. By varying three biologically relevant parameters—cell radius, cell division age and cell sensitivity to contact inhibition, we showed that tumor organoids with similar growth dynamics can express distinct morphologies and possess diverse cellular compositions. Taking advantage of the high-resolution of computational modeling, we applied the quantitative measures of compactness and accessible surface area, concepts that originated from the structural biology of proteins. Based on these analyses, we demonstrated that tumor organoids with similar sizes may differ in features associated with drug effectiveness, such as potential exposure to the drug or the extent of drug penetration. Both these characteristics might lead to major differences in tumor organoid’s response to therapy. This indicates that therapeutic protocols should not be based solely on tumor size, but take into account additional tumor features, such as their morphology or cellular packing density.

Geometry, packing, and evolutionary paths to increased multicellular size.

The evolutionary transition to multicellularity transformed life on earth, heralding the evolution of large, complex organisms. Recent experiments demonstrated that laboratory-evolved multicellular "snowflake yeast" readily overcome the physical barriers that limit cluster size by modifying cellular geometry [Jacobeen et al., Nat. Phys. 14, 286 (2018)10.1038/s41567-017-0002-y]. However, it is unclear why this route to large size is observed, rather than an evolved increase in intercellular bond strength. Here, we use a geometric model of the snowflake yeast growth form to examine the geometric efficiency of increasing size by modifying geometry and bond strength. We find that changing geometry is a far more efficient route to large size than evolving increased intercellular adhesion. In fact, increasing cellular aspect ratio is on average ∼13 times more effective than increasing bond strength at increasing the number of cells in a cluster. Modifying other geometric parameters, such as the geometric arrangement of mother and daughter cells, also had larger effects on cluster size than increasing bond strength. Simulations reveal that as cells reproduce, internal stress in the cluster increases rapidly; thus, increasing bond strength provides diminishing returns in cluster size. Conversely, as cells become more elongated, cellular packing density within the cluster decreases, which substantially decreases the rate of internal stress accumulation. This suggests that geometrically imposed physical constraints may have been a key early selective force guiding the emergence of multicellular complexity.

Penetration of anticancer drugs through tumour tissue as a function of cellular packing density and interstitial fluid pressure and its modification by bortezomib

BackgroundLimited penetration of anticancer drugs in solid tumours is a probable cause of drug resistance. Our previous results indicate that drug penetration depends on cellular packing density and adhesion between cancer cells.MethodsWe used epithelioid and round cell variants of the HCT-8 human colon carcinoma cell lines to generate tightly and loosely packed xenografts in nude mice. We measured packing density and interstitial fluid pressure (IFP) and studied the penetration of anti-cancer drugs through multilayered cell cultures (MCC) derived from epithelioid HCT-8 variants, and the distribution of doxorubicin in xenografts with and without pre-treatment with bortezomib.ResultsWe show lower packing density in xenografts established from round cell than epithelioid cell lines, with lower IFP in xenografts. There was better distribution of doxorubicin in xenografts grown from round cell variants, consistent with previous data in MCC. Bortezomib pre-treatment reduced cellular packing density, improved penetration, and enhanced cytotoxcity of several anticancer drugs in MCC derived from epithelioid cell lines. Pre-treatment of xenografts with bortezomib enhanced the distribution of doxorubicin within them.ConclusionsOur results provide a rationale for further investigation of agents that enhance the distribution of chemotherapeutic drugs in combination with conventional chemotherapy in solid tumours.

Solid stress inhibits the growth of multicellular tumor spheroids

In normal tissues, the processes of growth, remodeling, and morphogenesis are tightly regulated by the stress field; conversely, stress may be generated by these processes. We demonstrate that solid stress inhibits tumor growth in vitro, regardless of host species, tissue of origin, or differentiation state. The inhibiting stress for multicellular tumor spheroid growth in agarose matrices was 45 to 120 mm Hg. This stress, which greatly exceeds blood pressure in tumor vessels, is sufficient to induce the collapse of vascular or lymphatic vessels in tumors in vivo and can explain impaired blood flow, poor lymphatic drainage, and suboptimal drug delivery previously reported in solid tumors. The stress-induced growth inhibition of plateau-phase spheroids was accompanied, at the cellular level, by decreased apoptosis with no significant changes in proliferation. A concomitant increase in the cellular packing density was observed, which may prevent cells from undergoing apoptosis via a cell-volume or cell-shape transduction mechanism. These results suggest that solid stress controls tumor growth at both the macroscopic and cellular levels, and thus influences tumor progression and delivery of therapeutic agents.

Cellular expression of MAP 2 kinase in rat brain.

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density. Thus, MAP 2 kinase expression was particularly high in regions such as the locus coeruleus, the piriform cortex, the dentate gyrus granule cell layer, pyramidal cells of the hippocampus, the mitral cells of the olfactory bulb, and the large motor neurons of the V and VII nerves. This apparent ubiquitous distribution suggests an important role of MAP 2 kinase in the cellular functions in most cells of the adult brain.

Interrelationship among morphology, metabolism, and proliferation of tumor cells in monolayer and spheroid culture.

The proliferative activity of tumor cells is positively correlated with the cellular respiration rate. Thus, a decrease in the proliferative fraction during monolayer growth of EMT6 cells is associated with a decrease in the oxygen consumption rate both per cell and per cellular volume. These changes are paralleled by variations in the morphology of the tumor cells. The cellular volume, the cytoplasmic volume, the number and volume of mitochondria is decreased, whereas the volume of the nucleus remains relatively constant during the transition of cells from the exponential to the plateau growth phase. Similar observations have been made in outer, highly proliferative and inner non-proliferating cells in EMT6 spheroids. However, the local oxygen consumption in this three dimensional, tissue-like assemblage of cells is obviously modified by the cellular packing density leading to a rather uniform oxygen uptake in the proliferating and non-proliferating cell areas.

The stereotrode: A new technique for simultaneous isolation of several single units in the central nervous system from multiple unit records

A new method is described for the recording and discrimination of extracellular action potentials in CNS regions with high cellular packing density or where there is intrinsic variation in action potential amplitude during burst discharge. The method is based on the principle that cells with different ratios of distances from two electrode tips will have different spike-amplitude ratios when recorded on two channels. The two channel amplitude ratio will remain constant regardless of intrinsic variation in the absolute amplitude of the signals. The method has been applied to the rat hippocampal formation, from which up to 5 units have been simultaneously isolated. The construction of the electrodes is simple, relatively fast, and reliable, and their low tip impedances result in excellent signal to noise characteristics.

Immunohistochemical study of the development of serotonergic neurons in the rat CNS

In this study the development of serotonergic (5-HT) neurons is followed from their initiation of transmitter synthesis until the establishment of an essentially mature morphology. We have used the new and sensitive technique of 5-HT immunocytochemistry to visualize the precise features of this process. The great stability of this method, and the feasibility of counter-staining tissue sections permits the visualization of dendritic processes and axon terminals, as well as perikarya, and facilitates the localization of these structures with respect to non-5-HT components of the neuropil. Serial transverse and sagittal sections of rat fetuses on embryonic days (ED) 13, 14, 15, 17, 19 and 21, and postnatal rats on days 1,3,4 and 10 were examined. A detailed photomicrographic map showing the locations of 5-HT neurons at all prenatal stages is provided. The development of 5-HT neurons is evaluated in terms of their cellular morphology, particularly dendritic architecture, the relationship of these cells to the development of the surrounding brainstem, and the morphology and packing density of the 5-HT nuclei. From these considerations a model is proposed of the pattern of cell migration within the nuclei that give rise to the ascending 5-HT projections. At E14 a relatively simple configuration of bilateral superior (rostral) and inferior (caudal) 5-HT cell groups is present. In the period extending from E14 to E19 several subgroupings of these cells develop, presumably as the result of differential cell migration. Based on the predominant dendritic orientation of these cells it is possible to reconstruct their probable migratory paths. At E19 the 5-HT neurons are distributed in groups that are similar to those seen in the adult. In the time from E19 until the end of the first postnatal week there is rapid growth of 5-HT dendrites and a marked decrease in cellular packing density. These alterations shape the nuclear aggregates into the form seen in the adult. The development of the 5-HT cell groups is discussed in the context of known features of neurogenesis, migration, and axonal projections of the raphe and medial reticular nuclei of the brainstem. The possibility is raised that the decrease in cellular packing density in the 5-HT nuclei may reflect the appearance of the non-5-HT components of the raphe nuclei.

Morphological Alterations in the Medial Preoptic Area After Prenatal Administration of Phenobarbital

Pregnant Sabra mice received 3 g phenobarbital per kilogram milled food from gestational days (GD) 9 to 18 as their only food source; control females received milled food. All the females were divided into three groups and injected intraperitoneally with a single dose of 3H-thymidine on GD 13, 15 or 17. At 50 days of age of the offspring were perfused with 10% neutral formalin and their brains removed and prepared for autoradiography. Matching sagittal sections of the offspring were selected for the study of the medial preoptic area (MPOA) of the hypothalamus. The total number of cells per section was not significantly different between males and females, however, there was a greater cellular packing density (cell/mm2) in females (p less than 0.05). This sex difference disappeared in treated offspring, possibly due to a 76% decrease on GD 13 in the rate of cell proliferation in females (number of labeled cells but not in males (p less than 0.001); the greatest decrease in the number of labeled cells occurred in the sexually dimorphic areas of the MPOA (p less than 0.001). The area of the sagittal section of the MPOA did not differ significantly between sexes or treatments.

Stereological quantification of histological parameters in turpentine-induced hyperplasia of hamster cheek pouch epithelium

Epithelial hyperplasia was induced by regular painting with 50 per cent v/v turpentine in liquid paraffin. Point and intersect counting were used to analyse representative microscope fields photographed on black and white reversal film and examined on a back projector at a × 900 magnification. Nuclear-cytoplasmic ratio, nuclear and cellular packing density, nuclear and cell volume, epithelial thickness, epithelial volume, and volume to interface ratios of epithelium were estimated. Corrected numbers of nuclei and cells were derived using factors which made allowances for section thickness and nuclear size and assumed the nuclei to be approximately spherical. The results showed that both hypertrophy and hyperplasia occur in turpentine-treated hamster cheek pouch epithelium. At the histological level stereological techniques can be employed to detect quantitative similarities and differences between normal and pathological epithelium.

Stereological quantification of histological parameters in normal hamster cheek pouch epithelium

A stereological investigation was performed on histological sections of normal epithelium obtained from both cheek pouches of 5 hamsters. Point and intercept counting techniques were used to analyse representative microscope fields photographed on black and white reversal film and examined in a back projector at a viewing magnification of × 900. Cytological and histological parameters of defined epithelial components were estimated, including nuclear-cytoplasmic ratio, nuclear and cellular packing density, nuclear and cell volumes, epithelial thickness, epithelial volume and “volume to interface ratios” of epithelium. Epithelial thickness was also measured directly using a calibrated micrometer eyepiece. Numbers of nuclei and cells were obtained using correction factors which made allowances for section thickness and particle size and assumed the particles to be approximately spherical in shape. The results showed that it was possible to estimate some specific epithelial parameters stereologically at light microscope level.

Ontogeny of monoamine neurons in the locus coeruleus, raphe nuclei and substantia nigra of the rat

Synaptogenesis was studied in the monoamine (MA) cell groups locus coeruleus (LC), dorsal and medial raphe nuclei (RN) and substantia nigra, zona compacta (SN) between day 18 of gestation and postnatal day 60 using ethanolic phosphotungstic acid (E-PTA) to visualize synaptic profiles. Nuclear area, and cellular packing density (inversely proportional to area of neuropil) were also determined. As determined using the E-PTA method, synaptogenesis begins in the neuropil of the SN first, on or before 18 days of gestation, and in the LC and RN at 19 days. Synaptogenesis on MA cell perikarya is first observed in the SN, on or before 18 days, and in the LC and RN at 20 days. The onset of somatic synaptogenesis coincides with the beginning of nuclear growth and development of the neuropil (decrease in cellular packing density) in all MA cell areas, raising the possibility of common factors in the initiation of these processes. Nonsynaptic contacts precede the appearance of synaptic profiles both in the neuropil and on the somata of the MA cells of the LC, RN and SN, and may represent precursors of mature synapses or desmosome-like contacts. Somatosomatic nonsynaptic contacts occur only prenatally between adjacent MA neurons in the LC, RN and SN. Although some synaptogenesis occurs prenatally in these MA cell groups (indiciating that these parts of the MA circuitry may be functional before birth), most of this synaptogenesis occurs postnatally and continues into adulthood. Since such synaptogenesis does not begin until 2-4 days prior to birth, whereas these neurons and their processes exhibit MA fluorescence as early as 12-14 days of gestation, they apparently are capable of synthesizing transmitter and proliferating terminals before they themselves are innervated.