Spatial Characterization of Tumor-Infiltrating Lymphocytes and Breast Cancer Progression

Tumor-infiltrating lymphocytes (TILs) have been established as a robust prognostic biomarker in breast cancer, with emerging utility in predicting treatment response in the adjuvant and neoadjuvant settings. In this study, the role of TILs in predicting overall survival and progression-free interval was evaluated in two independent cohorts of breast cancer from the Cancer Genome Atlas (TCGA BRCA) and the Carolina Breast Cancer Study (UNC CBCS). We utilized machine learning and computer vision algorithms to characterize TIL infiltrates in digital whole-slide images (WSIs) of breast cancer stained with hematoxylin and eosin (H&E). Multiple parameters were used to characterize the global abundance and spatial features of TIL infiltrates. Univariate and multivariate analyses show that large aggregates of peritumoral and intratumoral TILs (forests) were associated with longer survival, whereas the absence of intratumoral TILs (deserts) is associated with increased risk of recurrence. Patients with two or more high-risk spatial features were associated with significantly shorter progression-free interval (PFI). This study demonstrates the practical utility of Pathomics in evaluating the clinical significance of the abundance and spatial patterns of distribution of TIL infiltrates as important biomarkers in breast cancer

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Hyperbaric Oxygen Promotes Proximal Bone Regeneration and Organized Collagen Composition during Digit Regeneration

<div><p>Oxygen is critical for optimal bone regeneration. While axolotls and salamanders have retained the ability to regenerate whole limbs, mammalian regeneration is restricted to the distal tip of the digit (P3) in mice, primates, and humans. Our previous study revealed the oxygen microenvironment during regeneration is dynamic and temporally influential in building and degrading bone. Given that regeneration is dependent on a dynamic and changing oxygen environment, a better understanding of the effects of oxygen during wounding, scarring, and regeneration, and better ways to artificially generate both hypoxic and oxygen replete microenvironments are essential to promote regeneration beyond wounding or scarring. To explore the influence of increased oxygen on digit regeneration <i>in vivo</i> daily treatments of hyperbaric oxygen were administered to mice during all phases of the entire regenerative process. Micro-Computed Tomography (μCT) and histological analysis showed that the daily application of hyperbaric oxygen elicited the same enhanced bone degradation response as two individual pulses of oxygen applied during the blastema phase. We expand past these findings to show histologically that the continuous application of hyperbaric oxygen during digit regeneration results in delayed blastema formation at a much more proximal location after amputation, and the deposition of better organized collagen fibers during bone formation. The application of sustained hyperbaric oxygen also delays wound closure and enhances bone degradation after digit amputation. Thus, hyperbaric oxygen shows the potential for positive influential control on the various phases of an epimorphic regenerative response.</p></div

Spatial Characterization of Tumor-Infiltrating Lymphocytes and Breast Cancer Progression

Tumor-infiltrating lymphocytes (TILs) have been established as a robust prognostic biomarker in breast cancer, with emerging utility in predicting treatment response in the adjuvant and neoadjuvant settings. In this study, the role of TILs in predicting overall survival and progression-free interval was evaluated in two independent cohorts of breast cancer from the Cancer Genome Atlas (TCGA BRCA) and the Carolina Breast Cancer Study (UNC CBCS). We utilized machine learning and computer vision algorithms to characterize TIL infiltrates in digital whole-slide images (WSIs) of breast cancer stained with hematoxylin and eosin (H&amp;E). Multiple parameters were used to characterize the global abundance and spatial features of TIL infiltrates. Univariate and multivariate analyses show that large aggregates of peritumoral and intratumoral TILs (forests) were associated with longer survival, whereas the absence of intratumoral TILs (deserts) is associated with increased risk of recurrence. Patients with two or more high-risk spatial features were associated with significantly shorter progression-free interval (PFI). This study demonstrates the practical utility of Pathomics in evaluating the clinical significance of the abundance and spatial patterns of distribution of TIL infiltrates as important biomarkers in breast cancer

HBO treatment delays wound closure and blastema formation.

<p>H&E staining of 7, 10, and 14 DPA sections of control (A-C) and HBO-treated (A'-C') digits show cellular changes in the marrow and delayed wound closure and blastema formation in HBO-treated digits. Arrows indicate areas of vasculature on control 7 DPA (A) and HBO treated 7 DPA (A') samples. (B') Epidermis remains open at 10 DPA in samples treated with hyperbaric oxygen. (C') Blastema formation is delayed until 14 DPA in HBO-treated samples when control samples (C) are already forming bone. Higher magnification analysis of the proximal endosteum show activated osteoblasts in HBO-treated samples (A'-C') while control samples return to an inactivated morphology at 10 DPA (B). Scale bars = 100 μm in panels and 5 μm in insets. (N = 8 HBO and N = 7 control with representative figure shown).</p

HBO treatment alters the morphology of regenerated bone.

<p>Polarized light micrographs of control and HBO treated digits at 7, 10, 14, and 21 DPA show differences in collagen fiber composition at distinct time points during regeneration. Collagen fiber alignment and thickness is readily identifiable under polarized light. Thinner fibers (a general indication of either thin collagen I fibrils, <50 nm in width, or collagen III fibers) appear green. Thick, more mature and aligned collagen I fibers appear red or yellow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#pone.0140156.ref022" target="_blank">22</a>]. Photomicrographs are presented in transmitted light (A—F) and polarized light (A'-F'). Digit orientation is portrayed by black cartoon on the upper right corner of each image. Scale bars = 100 μm. N = 3 with representative sample shown.</p

Continuous HBO treatment enhances bone degradation.

<p>(A) Bone regeneration during HBO application shows a similar rate of bone degradation before beginning to grow bone at 12 DPA (N = 4 mice, N = 16 digits). Samples were analyzed for bone growth using μCT. Data are normalized to initial 0 DPA bone volume and analyzed using a SS ANOVA algorithm accounting for variation between the individual mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#sec006" target="_blank">methods</a>). (B) Representative X-ray images of a regenerating P3 digit at 0, 7, 10, 17, and 28 DPA. (C) 14 DPA time point capture showing the P3 regenerative response in a control digit, (from A). (D) 14 DPA time point capture of a representative digit (from A) treated with continuous HBO twice daily showing increased bone degradation. Full time lapse sequence available in supplemental materials. (E) Effect of daily HBO application on osteoclast numbers at 7, 10, and 14 DPA. Results are expressed as mean ± SEM. ^# P<0.05, comparison of control to HBO. NOc/BPm; number of osteoclasts/bone perimeter. (F) Micro-CT scans of the same control sample seen in C and (G) same HBO sample seen in D are pseudo-colored according to trabecular thickness. (*) Asterisk indicates degradation through the proximal os-hole. Color changes indicate bone thickness in μm.</p

Hyperbaric oxygen extends the level of regenerative competency.

<p>A) Mallory trichrome staining at 14 and (B) 28 DPA of a HBO treated digit show proximal degradation of P3 bone and subsequent patterned bone and regeneration of the marrow cavity. Scale bar = 100 μm. Cross-sections of μCT-generated 3D renderings of the sample shown in 4B at 14 DPA (C) and 28 DPA (D) show regeneration of the marrow cavity and patterned distal outgrowth of bone. (E) μCT-generated 3D renderings show exacerbated bone degradation through the P3 joint area that regenerates along with the distal bone. The regenerated P3 shows a defect in the joint at DPA 37 (asterisk), which coincides with an imperfect joint lining in this area (G). (F) Mallory trichrome staining of the regenerated digit at 36 DPA shows imperfect joint patterning in the dorsal region of the joint (G, box 1 in F) when compared to a control P2/P3 joint (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#pone.0140156.s004" target="_blank">S4 Fig</a>). Scale bar = 100 μm. Higher magnification images show regenerated, but discontinuous staining of the cartilage joint region when compared to the non-degraded ventral region (H, box 2 in F). Scale bar = 25 μm. (I and J) Picro-Sirius red staining of control digits at 28 DPA shows regenerated woven bone, that (K) do not resolve trabecular woven bone to lamellar bone even after 77 days. Scale bar = 50 μm. (L) In contrast, 28 DPA HBO treated digits show parallel collagen I fibers that are more akin to the lamellar bone seen in the cortical bone stump. Scale bar = 50 μm. Original lamellar bone stump is outlined and indicated by an asterisk, highlighting the boundary with the newly regenerated bone. (N = 3, representative figure shown).</p